Method for Manufacturing Useful Substance

ABSTRACT

A method for producing an objective substance is provided. An objective substance is produced by culturing a microorganism which has been modified so that the activity of a dicarboxylic acid exporter protein is reduced in a medium, and collecting the objective substance from the medium.

This application is a Continuation of, and claims priority under 35U.S.C. §120 to, International Application No. PCT/JP2014/068368, filedJul. 9, 2014, and claims priority therethrough under 35 U.S.C. §119 toJapanese Patent Application No. 2013-144003, filed Jul. 9, 2013, andU.S. Provisional Patent Application No. 61/844,154, filed Jul. 9, 2013,the entireties of which are incorporated by reference herein. Also, theSequence Listing filed electronically herewith is hereby incorporated byreference (File name: 2016-01-07T_US-500_Seq_List; File size: 1032 KB;Date recorded: Jan. 7, 2016).

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to a method for producing a usefulsubstance using a microorganism.

2. Brief Description of the Related Art

When producing amino acids such as glutamic acid and alcohols such asisopropyl alcohol by fermentation, succinic acid is produced as one ofseveral by-products (WO2013/018734 and WO2008/114721). Consequently, theyield of the objective substance is decreased and, thus, not only thecost of the raw materials for the fermentation but also the cost ofpurifying the product are increased, which is uneconomical.

SUMMARY OF THE INVENTION

An aspect of the present invention is to develop a novel technique forimproving an objective substance-producing ability of a microorganismand thereby provide a method for efficiently producing an objectivesubstance.

The ability of a microorganism to produce an objective substance can beimproved by modifying the microorganism so that the activity of adicarboxylic acid exporter protein is reduced.

It is an aspect of the present invention to provide a method forproducing an objective substance, the method comprising:

culturing a microorganism having an objective substance-producingability in a medium to produce and accumulate the objective substance inthe medium or in cells of the microorganism; and

collecting the objective substance from the medium or the cells,

wherein the microorganism has been modified so that the activity of adicarboxylic acid exporter protein is reduced.

It is a further aspect or the present invention to provide the method asdescribed above, wherein the activity of the dicarboxylic acid exporterprotein is reduced by attenuating the expression of a gene encoding thedicarboxylic acid exporter protein or by deleting the gene.

It is a further aspect or the present invention to provide the method asdescribed above, wherein the gene encoding the dicarboxylic acidexporter protein is selected from the group consisting of yjjP, yjjB,yeeA, ynJM, sucE1, and combinations thereof.

It is a further aspect or the present invention to provide the method asdescribed above, wherein the yjjP gene is a DNA selected from the groupconsisting of:

(A) a DNA encoding a protein comprising the amino acid sequence of SEQID NO: 158 or 160;

(B) a DNA encoding a protein comprising the amino acid sequence of SEQID NO: 158 or 160, but including substitution, deletion, insertion, oraddition of one or several amino acid residues, the protein having anactivity to export a dicarboxylic acid;

(C) a DNA comprising the nucleotide sequence of SEQ ID NO: 157 or 159;and

(D) a DNA able to hybridize under stringent conditions with a nucleotidesequence complementary to the nucleotide sequence of SEQ ID NO: 157 or159, or with a probe that can be prepared from the complementarynucleotide sequence, and encoding a protein having an activity to exporta dicarboxylic acid.

It is a further aspect or the present invention to provide the method asdescribed above, wherein the yjjB gene is a DNA selected from the groupconsisting of:

(A) DNA encoding a protein comprising the amino acid sequence of SEQ IDNO: 162 or 164;

(B) DNA encoding a protein comprising the amino acid sequence of SEQ IDNO: 162 or 164, but including substitution, deletion, insertion, oraddition of one or several amino acid residues, the protein having anactivity to export a dicarboxylic acid;

(C) DNA comprising the nucleotide sequence of SEQ ID NO: 161 or 163; and

(D) DNA able to hybridize under stringent conditions with a nucleotidesequence complementary to the nucleotide sequence of SEQ ID NO: 161 or163, or with a probe that can be prepared from the complementarynucleotide sequence, and encoding a protein having an activity to exporta dicarboxylic acid.

It is a further aspect or the present invention to provide the method asdescribed above, wherein the yeeA gene is a DNA selected from the groupconsisting of:

(A) DNA encoding a protein comprising the amino acid sequence of SEQ IDNO: 166, 168, or 170;

(B) DNA encoding a protein comprising the amino acid sequence of SEQ IDNO: 166, 168, or 170, but including substitution, deletion, insertion,or addition of one or several amino acid residues, the protein having anactivity to export a dicarboxylic acid;

(C) DNA comprising the nucleotide sequence of SEQ ID NO: 165, 167, or169; and

(D) DNA able to hybridize under stringent conditions with a nucleotidesequence complementary to the nucleotide sequence of SEQ ID NO: 165,167, or 169, or with a probe that can be prepared from the complementarynucleotide sequence, and encoding a protein having an activity to exporta dicarboxylic acid.

It is a further aspect or the present invention to provide the method asdescribed above, wherein the ynfM gene is a DNA selected from the groupconsisting of:

(A) DNA encoding a protein comprising the amino acid sequence of SEQ IDNO: 172, 174, 176, 178, or 180;

(B) DNA encoding a protein comprising the amino acid sequence of SEQ IDNO: 172, 174, 176, 178, or 180, but including substitution, deletion,insertion, or addition of one or several amino acid residues, theprotein having an activity to export a dicarboxylic acid;

(C) DNA comprising the nucleotide sequence of SEQ ID NO: 171, 173, 175,177, or 179; and

(D) DNA able to hybridize under stringent conditions with a nucleotidesequence complementary to the nucleotide sequence of SEQ ID NO: 171,173, 175, 177, or 179 or with a probe that can be prepared from thecomplementary nucleotide sequence, and encoding a protein having anactivity to export a dicarboxylic acid.

It is a further aspect or the present invention to provide the method asdescribed above, wherein the sucE1 gene is a DNA selected from the groupconsisting of:

(A) DNA encoding a protein comprising the amino acid sequence of SEQ IDNO: 278 or 280;

(B) DNA encoding a protein comprising the amino acid sequence of SEQ IDNO: 278 or 280, but including substitution, deletion, insertion, oraddition of one or several amino acid residues, the protein having anactivity to export a dicarboxylic acid;

(C) DNA comprising the nucleotide sequence of SEQ ID NO: 277 or 279; and

(D) DNA able to hybridize under stringent conditions with a nucleotidesequence complementary to the nucleotide sequence of SEQ ID NO: 277 or279, or with a probe that can be prepared from the complementarynucleotide sequence, and encoding a protein having an activity to exporta dicarboxylic acid.

It is a further aspect or the present invention to provide the method asdescribed above, wherein the objective substance is a metabolite derivedfrom acetyl-CoA and/or an L-amino acid.

It is a further aspect or the present invention to provide the method asdescribed above, wherein the metabolite derived from acetyl-CoA and/orthe L-amino acid is selected from the group consisting of isopropylalcohol, ethanol, acetone, propylene, isoprene, 1,3-butanediol,1,4-butanediol, 1-propanol, 1,3-propanediol, 1,2-propanediol, ethyleneglycol, isobutanol, and combinations thereof.

It is a further aspect or the present invention to provide the method asdescribed above, wherein the metabolite derived from acetyl-CoA and/orthe L-amino acid is selected from the group consisting of citric acid,itaconic acid, acetic acid, butyric acid, 3-hydroxybutyric acid,polyhydroxybutyric acid, 3-hydroxyisobutyric acid, 3-aminoisobutyricacid, 2-hydroxyisobutyric acid, methacrylic acid, 6-aminocaproic acid,and combinations thereof.

It is a further aspect or the present invention to provide the method asdescribed above, wherein the metabolite derived from acetyl-CoA and/orthe L-amino acid is substances selected from the group consisting ofpolyglutamic acid, L-glutamic acid, L-glutamine, L-arginine,L-ornithine, L-citrulline, L-leucine, L-isoleucine, L-valine,L-cysteine, L-serine, L-proline, and combinations thereof.

It is a further aspect or the present invention to provide the method asdescribed above, wherein the L-glutamic acid is monoammonium L-glutamateor monosodium L-glutamate.

It is a further aspect or the present invention to provide the method asdescribed above, wherein the microorganism is a coryneform bacterium ora bacterium belonging to the family Enterobacteriaceae.

It is a further aspect or the present invention to provide the method asdescribed above, wherein the coryneform bacterium is Corynebacteriumglutamicum.

It is a further aspect or the present invention to provide the method asdescribed above, wherein the bacterium belonging to the familyEnterobacteriaceae is Escherichia coli, Pantoea ananatis, orEnterobacter aerogenes.

It is a further aspect or the present invention to provide the method asdescribed above, wherein the dicarboxylic acid is selected from thegroup consisting of malic acid, succinic acid, fumaric acid,2-hydroxyglutaric acid, and α-ketoglutaric acid.

It is a further aspect or the present invention to provide the method asdescribed above, wherein the microorganism has been further modified sothat malyl-CoA-producing ability is increased.

It is a further aspect or the present invention to provide the method asdescribed above, wherein the microorganism has been further modified sothat α-ketoglutarate synthase activity is increased.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 indicates the effect of reduced activities of dicarboxylic acidexporter proteins on the production of L-glutamic acid.

FIG. 2 indicates the effect of reduced activities of dicarboxylic acidexporter proteins on the yield of L-glutamic acid.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

Hereafter, the present invention will be explained in detail.

<1> Microorganism of the Present Invention

The microorganism can be a microorganism having an objectivesubstance-producing ability, which has been modified so that theactivity of a dicarboxylic acid exporter protein is reduced.

<1-1> Microorganism Having Objective Substance-Producing Ability

The term “objective substance” can be a compound such as acetyl-CoA,metabolites derived from acetyl-CoA (specifically, useful fermentationmetabolites derived from acetyl-CoA), and L-amino acids. Specificexamples of metabolites derived from acetyl-CoA and L-amino acidsinclude, for example, organic compounds such as isopropyl alcohol,ethanol, acetone, propylene, isoprene, 1,3-butanediol, 1-propanol,1,3-propanediol, 1,2-propanediol, ethylene glycol, and isobutanol;organic acids such as citric acid, itaconic acid, acetic acid, butyricacid, 3-hydroxybutyric acid, polyhydroxybutyric acid,3-hydroxyisobutyric acid, 3-aminoisobutyric acid, 2-hydroxyisobutyricacid, methacrylic acid, and 6-aminocaproic acid; and L-amino acids suchas polyglutamic acid, L-glutamic acid, L-glutamine, L-arginine,L-ornithine, L-citrulline, L-leucine, L-isoleucine, L-valine,L-cysteine, L-serine, and L-proline. Any amino acid can mean an L-aminoacid unless otherwise noted. One kind of objective substance may beproduced, or two or more kinds of objective substances may be produced.

The phrase “objective substance-producing ability” can mean an abilityto produce and accumulate an objective substance in cells or in a mediumwhen the microorganism is cultured in the medium, to such a degree thatthe objective substance can be collected from the cells or the medium.The microorganism having an objective substance-producing ability may bea microorganism that is able to accumulate the objective substance in amedium in an amount larger than that obtainable with a non-modifiedstrain. Moreover, the microorganism having an objectivesubstance-producing ability may also be a microorganism that is able toaccumulate the objective substance in an amount of 0.5 g/L or more, orin an amount of 1.0 g/L or more, in a medium. The objective substanceproduced by the microorganism may be one kind of substance or two ormore kinds of substances.

Examples of the microorganism include bacteria and yeast. Among them,bacteria are a particular example.

Examples of the bacteria include bacteria belonging to the familyEnterobacteriaceae and coryneform bacteria. Examples of the bacteriaalso include Alicyclobacillus bacteria and Bacillus bacteria.

Examples of bacteria belonging to the family Enterobacteriaceae includebacteria belonging to the genus Escherichia, Enterobacter, Pantoea,Klebsiella, Serratia, Erwinia, Photorhabdus, Providencia, Salmonella,Morganella, or the like. Specifically, bacteria classified into thefamily Enterobacteriaceae according to the taxonomy used in the NCBI(National Center for Biotechnology Information) database(http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?id=91347) canbe used.

The Escherichia bacteria are not particularly limited, and examplesthereof include those classified into the genus Escherichia according tothe taxonomy known to those skilled in the field of microbiology.Examples of the Escherichia bacteria include, for example, thosedescribed in the work of Neidhardt et al. (Backmann B. J., 1996,Derivations and Genotypes of some mutant derivatives of Escherichia coliK-12, pp. 2460-2488, Table 1, In F. D. Neidhardt (ed.), Escherichia coliand Salmonella Cellular and Molecular Biology/Second Edition, AmericanSociety for Microbiology Press, Washington, D.C.). Examples of theEscherichia bacteria include, for example, Escherichia coli. Specificexamples of Escherichia coli include, for example, Escherichia coliW3110 strain (ATCC 27325) and Escherichia coli MG1655 strain (ATCC47076), which are derived from the prototype wild-type strain K-12.

The Enterobacter bacteria are not particularly limited, and examplesinclude those classified into the genus Enterobacter according to thetaxonomy known to those skilled in the field of microbiology. Examplesthe Enterobacter bacterium include, for example, Enterobacteragglomerans and Enterobacter aerogenes. Specific examples ofEnterobacter agglomerans include, for example, the Enterobacteragglomerans ATCC 12287 strain. Specific examples of Enterobacteraerogenes include, for example, the Enterobacter aerogenes ATCC 13048strain, NBRC 12010 strain (Biotechnol. Bioeng., 2007, Mar. 27;98(2):340-348), and AJ110637 strain (FERM BP-10955). Examples theEnterobacter bacteria also include, for example, the strains describedin European Patent Application Laid-open (EP-A) No. 0952221. Inaddition, Enterobacter agglomerans also include some strains classifiedas Pantoea agglomerans.

The Pantoea bacteria are not particularly limited, and examples includethose classified into the genus Pantoea according to the taxonomy knownto those skilled in the field of microbiology. Examples the Pantoeabacteria include, for example, Pantoea ananatis, Pantoea stewartii,Pantoea agglomerans, and Pantoea citrea. Specific examples of Pantoeaananatis include, for example, the Pantoea ananatis LMG20103 strain,AJ13355 strain (FERM BP-6614), AJ13356 strain (FERM BP-6615), AJ13601strain (FERM BP-7207), SC17 strain (FERM BP-11091), and SC17(0) strain(VKPM B-9246). Some strains of Enterobacter agglomerans were recentlyreclassified into Pantoea agglomerans, Pantoea ananatis, Pantoeastewartii, or the like on the basis of nucleotide sequence analysis of16S rRNA etc. (Int. J. Syst. Bacteriol., 43, 162-173 (1993)). In thepresent invention, the Pantoea bacteria include those reclassified intothe genus Pantoea as described above.

Examples of the Erwinia bacteria include Erwinia amylovora and Erwiniacarotovora. Examples of the Klebsiella bacteria include Klebsiellaplanticola.

Examples of the coryneform bacteria include bacteria belonging to thegenus Corynebacterium, Brevibacterium, Microbacterium, or the like.

Specific examples of the coryneform bacteria include the followingspecies.

Corynebacterium acetoacidophilum

Corynebacterium acetoglutamicum

Corynebacterium alkanolyticum

Corynebacterium callunae

Corynebacterium glutamicum

Corynebacterium lilium

Corynebacterium melassecola

Corynebacterium thermoaminogenes (Corynebacterium efficiens)

Corynebacterium herculis

Brevibacterium divaricatum

Brevibacterium flavum

Brevibacterium immariophilum

Brevibacterium lactofermentum (Corynebacterium glutamicum)

Brevibacterium roseum

Brevibacterium saccharolyticum

Brevibacterium thiogenitalis

Corynebacterium ammoniagenes (Corynebacterium stationis)

Brevibacterium album

Brevibacterium cerinum

Microbacterium ammoniaphilum

Specific examples of the coryneform bacteria include the followingstrains.

Corynebacterium acetoacidophilum ATCC 13870

Corynebacterium acetoglutamicum ATCC 15806

Corynebacterium alkanolyticum ATCC 21511

Corynebacterium callunae ATCC 15991

Corynebacterium glutamicum ATCC 13020, ATCC 13032, ATCC 13060, ATCC13869, FERM BP-734

Corynebacterium lilium ATCC 15990

Corynebacterium melassecola ATCC 17965

Corynebacterium thermoaminogenes AJ12340 (FERM BP-1539)

Corynebacterium herculis ATCC 13868

Brevibacterium divaricatum ATCC 14020

Brevibacterium flavum ATCC 13826, ATCC 14067, AJ12418 (FERM BP-2205)

Brevibacterium immariophilum ATCC 14068

Brevibacterium lactofermentum ATCC 13869

Brevibacterium roseum ATCC 13825

Brevibacterium saccharolyticum ATCC 14066

Brevibacterium thiogenitalis ATCC 19240

Corynebacterium ammoniagenes (Corynebacterium stationis) ATCC 6871, ATCC6872

Brevibacterium album ATCC 15111

Brevibacterium cerinum ATCC 15112

Microbacterium ammoniaphilum ATCC 15354

The Corynebacterium bacteria include bacteria that had previously beenclassified into the genus Brevibacterium, but are presently united intothe genus Corynebacterium (Int. J. Syst. Bacteriol., 41, 255 (1991)).Moreover, Corynebacterium stationis includes bacteria that hadpreviously been classified as Corynebacterium ammoniagenes, but arepresently re-classified into Corynebacterium stationis on the basis ofnucleotide sequence analysis of 16S rRNA etc. (Int. J. Syst. Evol.Microbiol., 60, 874-879 (2010)).

The Bacillus bacteria are not particularly limited, and examples includethose classified into the genus Bacillus according to the taxonomy knownto those skilled in the field of microbiology. Examples the Bacillusbacteria include, for example, the following species.

Bacillus subtilis

Bacillus amyloliquefaciens

Bacillus pumilus

Bacillus licheniformis

Bacillus megaterium

Bacillus brevis

Bacillus polymixa

Bacillus stearothermophilus

Specific examples of Bacillus subtilis include, for example, Bacillussubtilis 168 Marburg strain (ATCC 6051) and Bacillus subtilis PY79(Plasmid, 1984, 12, 1-9). Specific examples of Bacillusamyloliquefaciens include, for example, Bacillus amyloliquefaciens Tstrain (ATCC 23842) and Bacillus amyloliquefaciens N strain (ATCC23845).

These strains are available from, for example, the American Type CultureCollection (Address: 12301 Parklawn Drive, Rockville, Md. 20852, P.O.Box 1549, Manassas, Va. 20108, United States of America). That is,registration numbers are given to the respective strains, and thestrains can be ordered by using these registration numbers (refer tohttp://www.atcc.org/). The registration numbers of the strains arelisted in the catalogue of the American Type Culture Collection.

The microorganism may be a bacterium inherently having an objectivesubstance-producing ability, or may be a microorganism modified so thatit has an objective substance-producing ability. The microorganismhaving an objective substance-producing ability can be obtained byimparting an objective substance-producing ability to such amicroorganism as mentioned above, or by enhancing an objectivesubstance-producing ability of such a microorganism as mentioned above.

To impart or enhance an objective substance-producing ability, methodsconventionally employed in the breeding of amino acid-producing strainsof coryneform bacteria, Escherichia bacteria, and so forth (see “AminoAcid Fermentation”, Gakkai Shuppan Center (Ltd.), 1st Edition, publishedMay 30, 1986, pp. 77-100) can be used. Examples of such methods include,for example, acquiring an auxotrophic mutant strain, acquiring anobjective substance analogue-resistant strain, acquiring a metabolicregulation mutant strain, and constructing a recombinant strain in whichthe activity of an objective substance biosynthetic enzyme is enhanced.In the breeding of objective substance-producing microorganisms, one ofthe above-described properties such as auxotrophy, analogue resistance,and metabolic regulation mutation may be imparted alone, or two or threeor more of such properties may be imparted in combination. Also, in thebreeding of objective substance-producing microorganisms, the activityof one of objective substance biosynthetic enzymes may be enhancedalone, or the activities of two or three or more of such enzymes may beenhanced in combination. Furthermore, imparting property(s) such asauxotrophy, analogue resistance, and metabolic regulation mutation canbe combined with enhancing the activity(s) of biosynthetic enzyme(s).

An auxotrophic mutant strain, objective substance analogue-resistantstrain, or metabolic regulation mutant strain having an objectivesubstance-producing ability can be obtained by subjecting a parentstrain or wild-type strain to a usual mutagenesis treatment, and thenselecting a strain exhibiting autotrophy, analogue resistance, or ametabolic regulation mutation, and having an objectivesubstance-producing ability from the obtained mutant strains. Examplesof the usual mutagenesis treatment include irradiation of X-ray orultraviolet and a treatment with a mutation agent such asN-methyl-N′-nitro-N-nitrosoguanidine (MNNG), ethyl methanesulfonate(EMS), and methyl methanesulfonate (MMS).

An objective substance-producing ability can also be imparted orenhanced by enhancing the activity of an enzyme involved in biosynthesisof an objective substance. An enzyme activity can be enhanced by, forexample, modifying a microorganism so that the expression of a genecoding for the enzyme is enhanced. Methods for enhancing gene expressionare described in WO00/18935, EP 1010755 A, and so forth. The detailedprocedures for enhancing enzyme activity will be described later.

Furthermore, an objective substance-producing ability can also beimparted or enhanced by reducing the activity of an enzyme thatcatalyzes a reaction branching away from the biosynthetic pathway of theobjective substance to generate a compound other than the objectivesubstance. Such enzymes can include an enzyme involved in decompositionof the objective substance. The method for reducing enzyme activity willbe described later.

Hereafter, objective substance-producing microorganisms and methods forimparting or enhancing an objective substance-producing ability will bespecifically exemplified. All of the properties of the objectivesubstance-producing microorganisms and modifications for imparting orenhancing an objective substance-producing ability may be usedindependently or in any appropriate combination.

<L-Glutamic Acid-Producing Microorganisms>

Examples of methods for imparting or enhancing L-glutamic acid-producingability include, for example, a method of modifying a microorganism sothat the microorganism has an increased activity or activities of one ormore kinds of enzymes such as the L-glutamic acid biosynthesis enzymes.Examples of such enzymes include, but are not particularly limited to,glutamate dehydrogenase (gdhA), glutamine synthetase (glnA), glutamatesynthetase (gltBD), isocitrate dehydrogenase (icdA), aconitate hydratase(acnA, acnB), citrate synthase (gltA), methylcitrate synthase (prpC),phosphoenolpyruvate carboxylase (ppc), pyruvate carboxylase (pyc),pyruvate kinase (pykA, pykF), pyruvate dehydrogenase (aceEF, lpdA),phosphoenolpyruvate synthase (ppsA), enolase (eno), phosphoglyceromutase(pgmA, pgmI), phosphoglycerate kinase (pgk), glyceraldehyde-3-phophatedehydrogenase (gapA), triose phosphate isomerase (tpiA), fructosebisphosphate aldolase (fbp), phosphofructokinase (pfkA, pfkB), glucosephosphate isomerase (pgi), 6-phosphogluconate dehydratase (edd),2-keto-3-deoxy-6-phosphogluconate aldolase (eda), transhydrogenase, andso forth. Shown in the parentheses after the names of the enzymes arethe names of the genes thereof (the same shall apply hereafter).Particular examples can include, for example, glutamate dehydrogenase,citrate synthase, phosphoenol pyruvate carboxylase, and methylcitratesynthase, among these enzymes.

Examples of strains belonging to the family Enterobacteriaceae andmodified so that the expression of the citrate synthase gene,phosphoenolpyruvate carboxylase gene, and/or glutamate dehydrogenasegene are increased include those disclosed in EP 1078989 A, EP 955368 A,and EP 952221 A. Furthermore, examples of strains belonging to thefamily Enterobacteriaceae and modified so that the expression of a geneof the Entner-Doudoroff pathway (edd, eda) is increased include thosedisclosed in EP 1352966 B. Also, examples of coryneform bacteriamodified so that expression of the glutamate synthetase gene (gltBD) isincreased include those disclosed in WO99/07853.

Examples of methods for imparting or enhancing L-glutamic acid-producingability also include, for example, a method of modifying a microorganismso that the microorganism has a reduced activity or activities of one ormore kinds of enzymes that catalyze a reaction branching away from thebiosynthesis pathway of L-glutamine to generate a compound other thanL-glutamic acid. Examples of such enzymes include, but are notparticularly limited to, α-ketoglutarate dehydrogenase (sucA, odhA),succinate dehydrogenase (sdhABCD), phosphotransacetylase (pta), acetatekinase (ack), acetohydroxy acid synthase (ilvG), acetolactate synthase(ilvI etc), formate acetyltransferase (pfl), lactate dehydrogenase(ldh), alcohol dehydrogenase (adh), glutamate decarboxylase (gadAB),1-pyroline-5-carboxylate dehydrogenase (putA), and so forth. deleteparticular example is, for example, the α-ketoglutarate dehydrogenaseactivity.

Methods for deleting or reducing the α-ketoglutarate dehydrogenaseactivity of Escherichia bacteria are disclosed in Japanese PatentLaid-open (Kokai) Nos. h5-244970 and h7-203980 etc. Furthermore, methodsfor deleting or reducing the α-ketoglutarate dehydrogenase activity ofCoryneform bacteria are disclosed in WO95/34672. Methods for deleting orreducing the α-ketoglutarate dehydrogenase activity ofEnterobacteriaceae bacteria such as Pantoea bacteria, Enterobacterbacteria, Klebsiella bacteria, and Erwinia bacteria are disclosed inU.S. Pat. Nos. 6,197,559, 6,682,912, 6,331,419, 8,129,151, andWO2008/075483. Methods for deleting or reducing the α-ketoglutaratedehydrogenase activity of Coryneform bacteria and Pantoea bacteria aredisclosed in WO2008/075483.

For example, in order to reduce the α-ketoglutarate dehydrogenaseactivity, sucA (odhA) gene encoding the E1o subunit of the enzyme can bemodified. Examples of strains in which the α-ketoglutarate dehydrogenaseactivity is reduced include, for example, the following strains.

Brevibacterium lactofermentum ΔS strain (WO95/34672)

Brevibacterium lactofermentum AJ12821 (FERM BP-4172, French Patent No.9401748)

Brevibacterium flavum AJ12822 (FERM BP-4173, French Patent No. 9401748)

Corynebacterium glutamicum AJ12823 (FERM BP-4174, French Patent No.9401748)

Corynebacterium glutamicum ATCC13869, OAGN, OA2-2, and OAGN2-2(WO2006/028298)

E. coli W3110sucA::Km^(r)

E. coli AJ12624 (FERM BP-3853)

E. coli AJ12628 (FERM BP-3854)

E. coli AJ12949 (FERM BP-4881)

Brevibacterium lactofermentum ΔS strain (WO95/34672)

Pantoea ananatis AJ13601 (FERM BP-7207, EP1078989)

Pantoea ananatis AJ13356 (FERM BP-6615, U.S. Pat. No. 6,331,419)

Pantoea ananatis SC17sucA (FERM BP-8646, WO2005/085419)

Klebsiella planticola AJ13410 (FERM BP-6617, U.S. Pat. No. 6,197,559)

Examples of L-glutamic acid-producing bacteria and parent strains whichcan be used to derive them also include the Pantoea ananatis AJ13355strain (FERM BP-6614), Pantoea ananatis SC17 strain (FERM BP-11091), andPantoea ananatis SC17(0) strain (VKPM B-9246). The AJ13355 strain is astrain isolated from soil in Iwata-shi, Shizuoka-ken, Japan as a strainthat can proliferate in a low pH medium containing L-glutamic acid and acarbon source. The SC17 strain is a strain selected as a lowphlegm-producing mutant strain from the AJ13355 strain (U.S. Pat. No.6,596,517). The SC17 strain was deposited at the independentadministrative agency, National Institute of Advanced Industrial Scienceand Technology, International Patent Organism Depository (currentlyindependent administrative agency, National Institute of Technology andEvaluation, International Patent Organism Depositary, #120, 2-5-8Kazusakamatari, Kisarazu-shi, Chiba-ken, 292-0818, Japan) on Feb. 4,2009, and assigned an accession number of FERM BP-11091. The AJ13355strain was deposited at the National Institute of Bioscience and HumanTechnology, Agency of Industrial Science and Technology (currently,independent administrative agency, National Institute of Technology andEvaluation, International Patent Organism Depositary, #120, 2-5-8Kazusakamatari, Kisarazu-shi, Chiba-ken, 292-0818, Japan) on Feb. 19,1998 and assigned an accession number of FERM P-16644. Then, the depositwas converted to an international deposit under the provisions ofBudapest Treaty on Jan. 11, 1999, and assigned an accession number ofFERM BP-6614.

Furthermore, examples of L-glutamic acid-producing bacteria and parentstrains which can be used to derive them also include Pantoea bacteriadeficient in the α-ketoglutarate dehydrogenase activity or having areduced α-ketoglutarate dehydrogenase activity. Examples of such strainsinclude the AJ13356 strain (U.S. Pat. No. 6,331,419), which is anα-KGDH-E1 subunit (sucA) gene-deficient strain of the AJ13355 strain,and the SC17sucA strain (U.S. Pat. No. 6,596,517), which is a sucAgene-deficient strain of the SC17 strain. The AJ13356 strain wasdeposited at the National Institute of Bioscience and Human-Technology,Agency of Industrial Science and Technology (currently, independentadministrative agency, National Institute of Technology and Evaluation,International Patent Organism Depositary, #120, 2-5-8 Kazusakamatari,Kisarazu-shi, Chiba-ken, 292-0818, Japan) on Feb. 19, 1998, and assignedan accession number of FERM P-16645. Then, the deposit was convertedinto an international deposit under the provisions of the BudapestTreaty on Jan. 11, 1999, and assigned an accession number of FERMBP-6616. The SC17sucA strain was assigned a private number of AJ417, anddeposited at the National Institute of Advanced Industrial Science andTechnology, International Patent Organism Depositary (currently,independent administrative agency, National Institute of Technology andEvaluation, International Patent Organism Depositary, #120, 2-5-8Kazusakamatari, Kisarazu-shi, Chiba-ken, 292-0818, Japan) on Feb. 26,2004, under an accession number of FERM BP-08646.

The AJ13355 strain was identified as Enterobacter agglomerans when itwas isolated, but it was recently reclassified as Pantoea ananatis onthe basis of nucleotide sequencing of 16S rRNA and so forth. Therefore,although the AJ13355, AJ13356, and AJ13601 strains are deposited at theaforementioned depository as Enterobacter agglomerans, they are referredto as Pantoea ananatis in this specification.

Furthermore, examples of L-glutamic acid-producing bacteria and parentstrains which can be used to derive them also include Pantoea bacteriasuch as the SC17sucA/RSFCPG+pSTVCB strain, AJ13601 strain, NP106 strain,and NA1 strain. The SC17sucA/RSFCPG+pSTVCB strain was obtained byintroducing the plasmid RSFCPG containing the citrate synthase gene(gltA), phosphoenolpyruvate carboxylase gene (ppsA), and glutamatedehydrogenase gene (gdhA) derived from Escherichia coli, and the plasmidpSTVCB containing the citrate synthase gene (gltA) derived fromBrevibacterium lactofermentum, into the SC17sucA strain. The AJ13601strain includes the SC17sucA/RSFCPG+pSTVCB strain and was selected forit's resistance to a high concentration of L-glutamic acid at a low pH.The NP106 strain was obtained from the AJ13601 strain by curing theRSFCPG and pSTVCB plasmids. The AJ13601 strain was deposited at theNational Institute of Bioscience and Human Technology, Agency ofIndustrial Science and Technology (currently, independent administrativeagency, National Institute of Technology and Evaluation, InternationalPatent Organism Depositary, #120, 2-5-8 Kazusakamatari, Kisarazu-shi,Chiba-ken, 292-0818, Japan) on Aug. 18, 1999, and assigned an accessionnumber FERM P-17516. Then, the deposit was converted to an internationaldeposit under the provisions of the Budapest Treaty on Jul. 6, 2000, andassigned an accession number FERM BP-7207.

Examples of L-glutamic acid-producing bacteria and parent strains whichcan be used to derive them also include strains in which both theα-ketoglutarate dehydrogenase (sucA) activity and the succinatedehydrogenase (sdh) activity are reduced or deleted (Japanese PatentLaid-open (Kokai) No. 2010-041920). Specific examples of such strainsinclude, for example, the sucAsdhA double-deficient strain of Pantoeaananatis NA1 strain and Corynebacterium glutamicum 8L3GΔSDH strain,which is the odhAsdhA double-deficient strain of Corynebacteriumglutamicum ATCC 14067 (Japanese Patent Laid-open (Kokai) No.2010-041920).

Examples of L-glutamic acid-producing bacteria and parent strains whichcan be used to derive them also include auxotrophic mutant strains.Specific examples of auxotrophic mutant strains include, for example, E.coli VL334thrC⁺ (VKPM B-8961, EP 1172433). E. coli VL334 (VKPM B-1641)is an L-isoleucine and L-threonine auxotrophic strain having mutationsin the thrC and ilvA genes (U.S. Pat. No. 4,278,765). E. coli VL334thrC⁺is an L-isoleucine-auxotrophic L-glutamic acid-producing bacteriumobtained by introducing a wild-type allele of the thrC gene into theVL334 strain. The wild-type allele of the thrC gene was introduced bythe method of general transduction using a bacteriophage P1 grown on thewild-type E. coli K-12 strain (VKPM B-7) cells.

Examples of L-glutamic acid-producing bacteria and parent strains whichcan be used to derive them also include strains having resistance to anaspartic acid analogue. Such strains can also be deficient in theα-ketoglutarate dehydrogenase activity. Specific examples of strainshaving resistance to an aspartic acid analogue and deficient in theα-ketoglutarate dehydrogenase activity include, for example, E. coliAJ13199 (FERM BP-5807, U.S. Pat. No. 5,908,768), E. coli FFRM P-12379,which additionally has a lowered L-glutamic acid-decomposing ability(U.S. Pat. No. 5,393,671), and E. coli AJ13138 (FERM BP-5565, U.S. Pat.No. 6,110,714).

Examples of methods for imparting or enhancing L-glutamic acid-producingability also include a method of modifying a bacterium so that theD-xylulose-5-phosphate phosphoketolase activity and/or thefructose-6-phosphate phosphoketolase activity are/is enhanced (JapanesePatent Laid-open (Kohyo) No. 2008-509661). Either theD-xylulose-5-phosphate phosphoketolase activity or thefructose-6-phosphate phosphoketolase activity may be enhanced, or bothmay be enhanced. In this specification, D-xylulose-5-phosphatephosphoketolase and fructose-6-phosphate phosphoketolase may becollectively referred to as phosphoketolase.

The D-xylulose-5-phosphate phosphoketolase activity can mean an activityfor converting xylulose-5-phosphate into glycelaldehyde-3-phosphate andacetyl phosphate by consuming phosphoric acid to release one molecule ofH₂O. This activity can be measured by the method described by Goldberg,M. et al. (Methods Enzymol., 9, 515-520, 1996) or the method describedby L. Meile (J. Bacteriol., 183:2929-2936, 2001).

The fructose-6-phosphate phosphoketolase activity can mean an activityfor converting fructose-6-phosphate into erythrose-4-phosphate andacetyl phosphate by consuming phosphoric acid to release one molecule ofH₂O. This activity can be measured by the method described by Racker, E.(Methods Enzymol., 5, 276-280, 1962) or the method described by L. Meile(J. Bacteriol., 183:2929-2936, 2001).

Examples of methods for imparting or enhancing L-glutamine-producingability can also include, for example, a method of amplifying the yhfKgene (WO2005/085419) or the ybjL gene (WO2008/133161), which is anL-glutamic acid secretion gene.

Furthermore, examples of methods for imparting or enhancing L-glutamicacid-producing ability to or in coryneform bacteria also include byimparting resistance to an organic acid analogue, respiratory inhibitor,or the like, and by imparting sensitivity to a cell wall synthesisinhibitor. Specific examples imparting monofluoroacetic acid resistance(Japanese Patent Laid-open (Kokai) No. 50-113209), imparting adenineresistance or thymine resistance (Japanese Patent Laid-open (Kokai) No.57-065198), attenuating the urease activity (Japanese Patent Laid-open(Kokai) No. 52-038088), imparting malonic acid resistance (JapanesePatent Laid-open (Kokai) No. 52-038088), imparting resistance tobenzopyrones or naphthoquinones (Japanese Patent Laid-open (Kokai) No.56-1889), imparting HOQNO resistance (Japanese Patent Laid-open (Kokai)No. 56-140895), imparting α-ketomalonic acid resistance (Japanese PatentLaid-open (Kokai) No. 57-2689), imparting guanidine resistance (JapanesePatent Laid-open (Kokai) No. 56-35981), imparting sensitivity topenicillin (Japanese Patent Laid-open (Kokai) No. 4-88994), and soforth.

Specific examples of such resistant bacteria include the followingstrains.

Brevibacterium flavum AJ3949 (FERM BP-2632, refer to Japanese PatentLaid-open (Kokai) No. 50-113209)

Corynebacterium glutamicum AJ11628 (FERM P-5736, refer to JapanesePatent Laid-open (Kokai) No. 57-065198)

Brevibacterium flavum AJ11355 (FERM P-5007, refer to Japanese PatentLaid-open (Kokai) No. 56-1889)

Corynebacterium glutamicum AJ11368 (FERM P-5020, refer to JapanesePatent Laid-open (Kokai) No. 56-1889)

Brevibacterium flavum AJ11217 (FERM P-4318, refer to Japanese PatentLaid-open (Kokai) No. 57-2869)

Corynebacterium glutamicum AJ11218 (FERM P-4319, refer to JapanesePatent Laid-open (Kokai) No. 57-2869)

Brevibacterium flavum AJ11564 (FERM BP-5472, refer to Japanese PatentLaid-open (Kokai) No. 56-140895)

Brevibacterium flavum AJ11439 (FERM BP-5136, refer to Japanese PatentLaid-open (Kokai) No. 56-35981)

Corynebacterium glutamicum H7684 (FERM BP-3004, refer to Japanese PatentLaid-open (Kokai) No. 04-88994)

Brevibacterium lactofermentum AJ11426 (FERM P-5123, refer to JapanesePatent Laid-open (Kokai) No. 56-048890)

Corynebacterium glutamicum AJ11440 (FERM P-5137, refer to JapanesePatent Laid-open (Kokai) No. 56-048890)

Brevibacterium lactofermentum AJ11796 (FERM P-6402, refer to JapanesePatent Laid-open (Kokai) No. 58-158192)

Furthermore, examples of methods for imparting or enhancing L-glutamicacid-producing ability to or in coryneform bacteria also include byenhancing expression of the yggB gene and introducing a mutant yggB genehaving a mutation in the coding region (WO2006/070944). The yggB gene isa gene coding for a mechanosensitive channel. The yggB gene of theCorynebacterium glutamicum ATCC 13032 strain corresponds to the sequencecomplementary to the sequence of the nucleotide numbers 1,336,091 to1,337,692 in the genome sequence registered as Genbank Accession No.NC_003450 in the NCBI database, and is also called NCgl1221. The YggBprotein is registered as GenBank accession No. NP_600492. The nucleotidesequence of the yggB gene of Corynebacterium glutamicum 2256 (ATCC13869) and the amino acid sequence of the YggB protein encoded by thegene are shown in SEQ ID NOS: 273 and 274, respectively.

Examples of the mutant yggB gene which can be used in the aforementionedmethods include yggB genes having the following mutation(s). The YggBprotein encoded by a mutant yggB gene is also referred to as a mutantYggB protein. A yggB gene not having such mutation(s) and the YggBprotein encoded by the gene are also referred to as a wild-type yggBgene and wild-type YggB protein, respectively. Examples of the wild-typeYggB protein include, for example, a protein having the amino acidsequence shown in SEQ ID NO: 274.

(1) Mutation on C-Terminal Side

The mutation on the C-terminal side is a mutation introduced into a partof the nucleotide sequence of the region coding for the sequence of theamino acid numbers 419 to 533 in SEQ ID NO: 274. Although the mutationon the C-terminal side is not particularly limited so long as a mutationis introduced into at least a part of the nucleotide sequence of theaforementioned region, the mutation on the C-terminal side can be amutation for inserting an insertion sequence (henceforth also referredto as “IS”) or inserting a transposon. The mutation on the C-terminalside may be any mutation that introduces an amino acid substitution(missense mutation), a mutation causing a frame shift induced byinsertion of the aforementioned IS, or the like, and that introduces anonsense mutation.

Examples of the mutation on the C-terminal side include, for example, amutation for inserting a nucleotide sequence at the site coding for thevaline residue at position 419 in the wild-type YggB protein (2A-1-typemutation). The 2A-1-type mutation may result in, for example, deletionor substitution of a part or the whole of amino acid residues atpositions 419 to 533 of the wild-type YggB protein. Specific examples ofthe mutant yggB gene having the 2A-1-type mutation include, for example,a yggB gene inserted with IS next to “G” at position 1255 in SEQ ID NO:273, and thereby coding for a mutant YggB protein of 423 amino residuesin the full length, which is shorter than the native YggB protein (SEQID NO: 274) (Japanese Patent Laid-open (Kokai) No. 2007-222163).

Examples of the mutation on the C-terminal side can also include amutation for replacing a proline residue present in positions 419 to 533in the wild-type YggB protein with another amino acid residue. Examplesof such a proline residue include those at positions 424, 437, 453, 457,462, 469, 484, 489, 497, 515, 529, and 533 in the wild-type YggBprotein.

(2) Mutation in Transmembrane Region

It is estimated that the YggB protein encoded by the yggB gene has fivetransmembrane regions. In the amino acid sequence of the wild-type YggBprotein of SEQ ID NO: 274, the transmembrane regions correspond to theregions of the amino acid numbers 1 to 23 (first transmembrane region),25 to 47 (second transmembrane region), 62 to 84 (third transmembraneregion), 86 to 108 (fourth transmembrane region), and 110 to 132 (fifthtransmembrane region). The yggB gene may have a mutation in any of thesetransmembrane regions. The mutation in the transmembrane region can be amutation that includes substitution, deletion, addition, insertion, orinversion of one or several amino acid residues that does not cause aframe shift mutation or nonsense mutation. Examples of the mutation inthe transmembrane region include a mutation for inserting one or severalamino acid residues (e.g. Cys-Ser-Leu) between the leucine residue atposition 14 and the tryptophan residue at position 15, a mutation forreplacing the alanine residue at position 100 with another amino acidresidue (e.g. an amino acid having a hydroxy group in the side chainthereof, i.e. Thr, Ser, or Tyr; preferably Thr), and a mutation forreplacing the alanine residue at position 111 with another amino acidresidue (e.g. an amino acid having a hydroxy group in the side chainthereof, i.e. Thr, Ser, or Tyr; preferably Thr), in the amino acidsequence shown in SEQ ID NO: 274, and so forth. Specific examples of themutant yggB gene having such a mutation in transmembrane region include,for example, a yggB gene having the sequence TTCATTGTG inserted next tothe “G” at position 44 in SEQ ID NO: 273 (A1-type mutation), a yggB genein which the “G” at position 298 in SEQ ID NO: 273 is replaced with an“A” (19-type mutation), and a yggB gene in which the “C” at position 332in SEQ ID NO: 273 is replaced with a “T” (L30-type mutation).

When the wild-type YggB protein has an amino acid sequence other thanthe amino acid sequence shown in SEQ ID NO: 274, the mutant yggB genemay have a mutation in a region coding for the amino acid residuecorresponding to the amino acid residue at the aforementioned positionin SEQ ID NO: 274. In an arbitrary wild-type YggB protein, the aminoacid residue that is “the amino acid residue corresponding to the aminoacid residue at the aforementioned position in SEQ ID NO: 274” can bedetermined based on an alignment between the amino acid sequence of thewild-type YggB protein and the amino acid sequence of SEQ ID NO: 274.

<L-Glutamine-Producing Microorganisms>

Examples of the method for imparting or enhancing L-glutamine-producingability include, for example, a method of modifying a bacterium so thatthe activity or activities of one or more kinds of enzymes such as theL-glutamine biosynthesis enzymes are enhanced. Examples of such enzymesinclude, but are not particularly limited to, glutamate dehydrogenase(gdhA) and glutamine synthetase (glnA). The glutamine synthetaseactivity can also be enhanced by disruption of the glutamineadenylyltransferase gene (glnE) or disruption of the PII control proteingene (glnB) (EP 1229121).

Examples of the method for imparting or enhancing L-glutamine-producingability also include, for example, a method of modifying a bacterium sothat the activity or activities of one or more kinds of enzymes thatcatalyze a reaction branching away from the biosynthesis pathway ofL-glutamine to generate a compound other than L-glutamine are reduced.Examples of such enzymes include, but not particularly limited to,glutaminase.

Examples of L-glutamine-producing bacteria and parent strains which canbe used to derive them include coryneform bacteria in which the activityor activities of glutamate dehydrogenase (gdhA) and/or glutaminesynthetase (glnA) (EP 1229121, EP 1424398) are enhanced, and coryneformbacteria in which the glutaminase activity (Japanese Patent Laid-open(Kokai) No. 2004-187684) is reduced. Also, examples of L-glutamicacid-producing bacteria and parent strains which can be used to derivethem include a strain belonging to the genus Escherichia and having amutant glutamine synthetase in which the tyrosine residue of theposition 397 is replaced with another amino acid residue (U.S. PatentPublished Application No. 2003/0148474).

Examples of the methods for imparting or enhancing L-glutamine-producingability to or in coryneform bacteria also include the method ofimparting 6-diazo-5-oxo-norleucine resistance (Japanese Patent Laid-open(Kokai) No. 3-232497), the method of imparting purine analogueresistance and methionine sulfoxide resistance (Japanese PatentLaid-open (Kokai) No. 61-202694), the method of imparting α-ketomalonicacid resistance (Japanese Patent Laid-open (Kokai) No. 56-151495), andso forth.

Specific examples of coryneform bacteria having L-glutamine-producingability include, for example, the following strains.

Brevibacterium flavum AJ11573 (FERM P-5492, Japanese Patent Laid-open(Kokai) No. 56-151495)

Brevibacterium flavum AJ11576 (FERM BP-10381, Japanese Patent Laid-open(Kokai) No. 56-151495)

Brevibacterium flavum AJ12212 (FERM P-8123, Japanese Patent Laid-open(Kokai) No. 61-202694)

<L-Proline-Producing Microorganisms>

Examples of L-proline-producing bacteria and parent strains which can beused to derive them include bacteria having γ-glutamyl kinasedesensitized to feedback inhibition by L-proline and bacteria in whichthe L-proline decomposition system is attenuated. A method for modifyinga bacterium by using a DNA encoding γ-glutamyl kinase desensitized tofeedback inhibition by L-proline is disclosed in the report of Dandekarand Uratsu (J. Bacteriol. 170, 12: 5943-5945 (1988)). Also, examples ofa method for obtaining a bacterium in which the L-proline decompositionsystem is attenuated include, for example, methods of introducing amutation into a proline dehydrogenase gene so that the activity of theencoded enzyme is reduced. Specific examples of bacteria havingL-proline-producing ability include E. coli NRRL B-12403 and NRRLB-12404 (British Patent No. 2075056), E. coli VKPM B-8012 (RussianPatent Application No. 2000124295), E. coli mutant strains having aplasmid described in German Patent No. 3127361, and E. coli mutantstrains having a plasmid described by Bloom F. R. et al. (The 15th Miamiwinter symposium, 1983, p. 34).

Also, specific examples of bacteria having L-proline-producing abilityinclude E. coli 702 strain (VKPM B-8011), which is a3,4-dehydroxyproline and azetidine-2-carboxylate resistant strain, E.coli 702ilvA strain (VKPM B-8012), which is an ilvA gene-deficientstrain of the 702 strain, and E. coli strains in which the activity of aprotein encoded by b2682, b2683, b1242, or b3434 gene (Japanese PatentLaid-open (Kokai) No. 2002-300874) is enhanced.

<L-Arginine-Producing Microorganisms>

Examples of methods for imparting or enhancing L-arginine-producingability include, for example, a method of modifying a microorganism sothat the microorganism has an increased activity or activities of one ormore kinds of enzymes such as the L-arginine biosynthesis enzymes.Examples of such enzymes include, but are not particularly limited to,N-acetylglutamate synthase (argA), N-acetylglutamyl phosphate reductase(argC), ornithine acetyl transferase (argJ), N-acetylglutamate kinase(argB), acetylomithine transaminase (argD), acetylomithine deacetylase(argE), ornithine carbamoyl transferase (argF), argininosuccinatesynthetase (argG), argininosuccinate lyase (argH), and carbamoylphosphate synthetase (carAB). As the N-acetylglutamate synthase gene(argA), for example, a gene encoding a mutant enzyme desensitized tofeedback inhibition by L-arginine by substitution for the amino acidsequence corresponding to the positions 15 to 19 of the wild-type enzyme(European Patent Laid-open No. 1170361) can be used.

Examples of a microorganism having an L-arginine-producing abilityinclude Escherichia coli mutant strains having resistance toa-methylmethionine, p-fluorophenylalanine, D-arginine, argininehydroxamate, S-(2-aminoethyl)-cysteine, α-methylserine,β-2-thienylalanine, or sulfaguanidine (refer to Japanese PatentLaid-open (Kokai) No. 56-106598), and so forth. Examples of amicroorganism having an L-arginine-producing ability also include the E.coli 237 strain (Russian Patent Application No. 2000117677), which is anL-arginine-producing bacterium harboring N-acetyl glutamate synthasehaving a mutation resistant to feedback inhibition by L-arginine andthereby having a high activity. The E. coli 237 strain was deposited atthe Russian National Collection of Industrial Microorganisms (VKPM, 1Dorozhny proezd., 1 Moscow 117545, Russia) on Apr. 10, 2000 under anaccession number of VKPM B-7925, and the deposit was converted to aninternational deposit under the provisions of Budapest Treaty on May 18,2001. Examples of a microorganism having L-arginine-producing abilityalso include the E. coli 382 strain (Japanese Patent Laid-open (Kokai)No. 2002-017342), which is an L-arginine-producing bacterium derivedfrom the 237 strain and having an improved acetic acid-assimilatingability. The E. coli 382 strain was deposited at the Russian NationalCollection of Industrial Microorganisms (VKPM, 1 Dorozhny proezd., 1Moscow 117545, Russia) on Apr. 10, 2000 under accession number of VKPMB-7926.

Examples of a microorganism having L-arginine-producing ability alsoinclude, coryneform bacterium wild-type strains; the coryneformbacterium strains having resistance to a drug such as sulfa drugs,2-thiazolealanine, and α-amino-β-hydroxy valerate; the coryneformbacterium strains having resistance to 2-thiazolealanine and furtherexhibiting auxotrophy for L-histidine, L-proline, L-threonine,L-isoleucine, L-methionine, or L-tryptophan (Japanese Patent Laid-open(Kokai) No. 54-44096); coryneform bacterium strains resistant toketomalonic acid, fluoromalonic acid, or monofluoroacetic acid (JapanesePatent Laid-open (Kokai) No. 57-18989); coryneform bacterium strainsresistant to argininol (Japanese Patent Publication No. 62-24075);coryneform bacterium strains resistant to X-guanidine (X represents analiphatic chain or a derivative thereof, Japanese Patent Laid-open(Kokai) No. 2-186995); and so forth. Examples of coryneform bacteriahaving L-arginine-producing ability also include mutant strainsresistant to 5-azauracil, 6-azauracil, 2-thiouracil, 5-fluorouracil,5-bromo-uracil, 5-azacytosine, 6-azacytosine, etc; mutant strainsresistant to arginine hydroxamate and 2-thiouracil; mutant strainsresistant to arginine hydroxamate and 6-azauracil (Japanese PatentLaid-open (Kokai) No. 49-126819); mutant strains resistant to histidineanalog or tryptophan analog (Japanese Patent Laid-open (Kokai) No.52-114092); mutant strains resistant to at least one of methionine,histidine, threonine, proline, isoleucine, lysine, adenine, guanine, anduracil (or uracil precursor) (Japanese Patent Laid-open (Kokai) No.52-99289); mutant strains resistant to arginine hydroxamate (Japanesepublished examined application No. 51-6754); mutant strains auxotrophicfor succinate or resistant to nucleic acid base analogs (Japanese PatentLaid-open (Kokai) No. 58-9692); mutant strains deficient in argininedecomposition ability, having resistant to arginine antagonist andcanavanine, and auxotrophic for lysine (Japanese Patent Laid-open(Kokai) No. 52-8729); mutant strains resistant to arginine, argininehydroxamate, homoarginine, D-arginine, and canavanine, or resistant toarginine hydroxamate and 6-azauracil (Japanese Patent Laid-open (Kokai)No. 53-143288); mutant strains resistant to canavanine (Japanese PatentLaid-open (Kokai) No. 53-3586), and so forth.

Specific examples of coryneform bacteria having L-arginine-producingability include the following strains.

Corynebacterium glutamicum (Brevibacterium flavum) AJ11169 (FERMBP-6892)

Brevibacterium lactofermentum AJ12092 (FERM BP-6906)

Brevibacterium flavum AJ11336 (FERM BP-6893)

Brevibacterium flavum AJ11345 (FERM BP-6894)

Brevibacterium lactofermentum AJ12430 (FERM BP-2228)

Examples of L-arginine-producing bacteria and parent strains which canbe used to derive them also include strains deficient in ArgR, which isan arginine repressor (U.S. Patent Published Application No.2002-0045223), and strains in which intracellular glutamine synthetaseactivity is increased (U.S. Patent Published Application No.2005-0014236).

<L-Citrulline-Producing Bacteria and L-Ornithine-ProducingMicroorganisms>

The biosynthetic pathways of L-citrulline and L-ornithine are common tothat of L-arginine. Therefore, an ability to produce L-citrulline and/orL-ornithine can be imparted or enhanced by increasing the activity oractivities of N-acetylglutamate synthase (argA), N-acetylglutamylphosphate reductase (argC), ornithine acetyltransferase (argJ),N-acetylglutamate kinase (argB), acetylornithine transaminase (argD),and/or acetylornithine deacetylase (argE) (WO2006/35831).

<L-Leucine-Producing Microorganisms>

Examples of methods for imparting or enhancing L-leucine-producingability include, for example, a method of modifying a microorganism sothat the microorganism has an increased activity or activities of one ormore kinds of enzymes such as the L-leucine biosynthesis enzymes.Examples of such enzymes include, but not particularly limited to, theenzymes encoded by the genes of the leuABCD operon. For enhancing theactivity of such an enzyme, for example, the mutant leuA gene coding foran isopropyl maleate synthase desensitized to feedback inhibition byL-leucine (U.S. Pat. No. 6,403,342) can be used.

Examples of L-leucine-producing bacteria and parent strains which can beused to derive them include, but are not limited to, strains belongingto the genus Escherichia, such as E. coli strains resistant to leucine(for example, the 57 strain (VKPM B-7386, U.S. Pat. No. 6,124,121)), E.coli strains resistant to an leucine analogue such asβ-2-thienylalanine, 3-hydroxyleucine, 4-azaleucine, and5,5,5-trifluoroleucine (Japanese Patent Publication (Kokoku) No.62-34397 and Japanese Patent Laid-open (Kokai) No. 8-70879), E. colistrains obtained by a gene engineering technique described inWO96/06926, and E. coli H-9068 (Japanese Patent Laid-open (Kokai) No.8-70879). Furthermore, examples of coryneform bacteria havingL-leucine-producing ability include Brevibacterium lactofermentum AJ3718(FERM P-2516), which is resistant to 2-thiazole alanine andβ-hydroxyleucine and auxotrophic for isoleucine and methionine.

<L-Isoleucine-Producing Microorganisms>

Examples of L-isoleucine-producing bacteria and parent strains which canbe used to derive them include, but are not limited to, mutant strainshaving resistance to 6-dimethylaminopurine (Japanese Patent Laid-open(Kokai) No. 5-304969), mutant strains having resistance to an isoleucineanalogue such as thiaisoleucine and isoleucine hydroxamate, and mutantstrains having resistance to such an isoleucine analogue and furtherhaving resistance to DL-ethionine and/or arginine hydroxamate (JapanesePatent Laid-open (Kokai) No. 5-130882). Also, a recombinant straintransformed with a gene encoding a protein involved in L-isoleucinebiosynthesis such as threonine deaminase and acetohydroxy acid synthasecan be used as a parent strain (Japanese Patent Laid-open (Kokai) No.2-458, FR 0356739, U.S. Pat. No. 5,998,178). Furthermore, examples ofcoryneform bacteria having L-isoleucine-producing ability include thecoryneform bacterium in which brnE gene coding for a branched chainamino acid excretion protein is amplified (Japanese Patent Laid-open(Kokai) No. 2001-169788), the coryneform bacterium to whichL-isoleucine-producing ability is imparted by protoplast fusion with anL-lysine-producing bacterium (Japanese Patent Laid-open (Kokai) No.62-74293), the coryneform bacterium in which homoserine dehydrogenase isenhanced (Japanese Patent Laid-open (Kokai) No. 62-91193), the threoninehydroxamate resistant strain (Japanese Patent Laid-open (Kokai) No62-195293), the α-ketomalonic acid resistant strain (Japanese PatentLaid-open (Kokai) No. 61-15695), the methyllysine resistant strain(Japanese Patent Laid-open (Kokai) No. 61-15696), and Brevibacteriumflavum AJ12149 (FERM BP-759, U.S. Pat. No. 4,656,135).

<L-Valine-Producing Microorganisms>

Examples of methods for imparting or enhancing L-valine-producingability include, for example, a method of modifying a microorganism sothat the microorganism has an increased activity or activities of one ormore kinds of enzymes such as the L-valine biosynthesis enzymes.Examples of such enzymes include, but are not particularly limited to,the enzymes encoded by the genes of the ilvGMEDA operon. Expression ofthe ilvGMEDA operon is suppressed (attenuated) by L-valine,L-isoleucine, and/or L-leucine. Therefore, to enhance the activity ofsuch an enzyme, suppressing the expression by the produced L-valine isinhibited by removing or modifying a region required for theattenuation. Furthermore, the threonine deaminase encoded by the ilvAgene is an enzyme that catalyzes the deamination reaction of L-threonineresulting 2-ketobutyric acid, which is the rate-limiting step of theL-isoleucine biosynthesis system. Therefore, for L-valine production,the ilvA gene can be, for example, disrupted, and thereby the threoninedeaminase activity is decreased.

Specific examples of L-valine-producing bacteria and parent strainswhich can be used to derive them include, for example, E. coli strainsmodified so as to overexpress the ilvGMEDA operon (U.S. Pat. No.5,998,178).

Examples of L-valine-producing bacteria and parent strains which can beused to derive them also include mutant strains having a mutation inamino-acyl t-RNA synthetase (U.S. Pat. No. 5,658,766). Examples of suchstrains include, for example, E. coli VL1970, which has a mutation inthe ileS gene encoding isoleucine t-RNA synthetase. E. coli VL1970 wasdeposited at the Russian National Collection of IndustrialMicroorganisms (VKPM, 1 Dorozhny Proezd, 1 Moscow 117545, Russia) onJun. 24, 1988 under the accession number of VKPM B-4411. Examples ofL-valine-producing bacteria and parent strains which can be used toderive them also include mutant strains requiring lipoic acid for growthand/or lacking H⁺-ATPase (WO96/06926).

Examples of coryneform bacteria having L-valine-producing abilityinclude, for example, strains modified so that the expression of geneencoding an enzyme involved in biosynthesis of L-valine is increased.Examples of the enzyme involved in biosynthesis of L-valine include, forexample, enzymes encoded by ilvBNC operon, i.e. acetohydroxy acidsynthase encoded by ilvBN and isomeroreductase encoded by ilvC(WO00/50624). Because the ilvBNC operon is subject to expressionregulation of the operon by L-valine, L-isoleucine, and/or L-leucine, itis preferred that the attenuation is inhibited in order to inhibit thesuppression of expression by the produced L-valine.

Examples of coryneform bacteria having L-valine-producing abilityinclude, for example, strains modified so that the activity oractivities of one or more kinds of enzymes such as enzymes involved inmetabolic pathway that decreases L-valine production are reduced.Examples of such enzymes include, for example, threonine dehydrataseinvolved in the L-leucine synthesis, and the enzymes involved in theD-pantothenic acid synthesis (WO00/50624).

Examples of L-valine-producing bacteria and parent strains which can beused to derive them also include strains resistant to an amino acidanalogue or the like. Examples of such strains include, for example, thecoryneform bacterium strains which are auxotrophic for L-isoleucine andL-methionine, and resistant to D-ribose, purine ribonucleoside, orpyrimidine ribonucleoside, and have an ability to produce L-valine (FERMP-1841, FERM P-29) (Japanese Patent Publication No. 53-025034),coryneform bacterium strains resistant to polyketides (FERM P-1763, FERMP-1764) (Japanese Patent Publication No. 06-065314), and coryneformbacterium strains resistant to L-valine in a medium containing aceticacid as the sole carbon source and sensitive to pyruvic acid analogues(fluoropyruvic acid etc.) in a medium containing glucose as the solecarbon source (FERM BP-3006, BP-3007) (Japanese Patent No. 3006929).

<L-Cysteine-Producing Microorganisms>

Examples of methods for imparting or enhancing L-cysteine-producingability include, for example, a method of modifying a bacterium so thatthe bacterium has an increased activity or activities of one or morekinds of enzymes such as the L-cysteine biosynthesis enzymes. Examplesof such enzymes include, but are not particularly limited to, serineacetyltransferase (cysE) and 3-phosphoglycerate dehydrogenase (serA).The serine acetyltransferase activity can be enhanced by, for example,introducing a mutant cysE gene coding for a mutant serineacetyltransferase resistant to feedback inhibition by cysteine into abacterium. Such a mutant serine acetyltransferase is disclosed in, forexample, Japanese Patent Laid-open (Kokai) No. 11-155571 and U.S. PatentPublished Application No. 20050112731. Furthermore, the3-phosphoglycerate dehydrogenase activity can be enhanced by, forexample, introducing a mutant serA gene coding for a mutant3-phosphoglycerate dehydrogenase resistant to feedback inhibition byserine into a bacterium. Such a mutant 3-phosphoglycerate dehydrogenaseis disclosed in, for example, U.S. Pat. No. 6,180,373.

Furthermore, examples of methods for imparting or enhancingL-cysteine-producing ability also include, for example, a method ofmodifying a bacterium so that the bacterium has a reduced activity oractivities of one or more kinds of enzymes such as the enzymes thatcatalyze a reaction branching away from the biosynthesis pathway ofL-cysteine to generate a compound other than L-cysteine. Examples ofsuch enzymes include, for example, enzymes involved in decomposition ofL-cysteine. Examples of the enzymes involved in decomposition ofL-cysteine include, but are not particularly limited to,cystathionine-β-lyase (metC, Japanese Patent Laid-open (Kokai) No.11-155571; Chandra et al., Biochemistry, 21 (1982) 3064-3069),tryptophanase (tnaA, Japanese Patent Laid-open (Kokai) No. 2003-169668;Austin Newton et al., J. Biol. Chem., 240 (1965) 1211-1218),O-acetylserine sulfhydrylase B (cysM, Japanese Patent Laid-open (Kokai)No. 2005-245311), the malY gene product (Japanese Patent Laid-open(Kokai) No. 2005-245311), the d0191 gene product of Pantoea ananatis(Japanese Patent Laid-open (Kokai) No. 2009-232844), and cysteinedesulfhydrase (aecD) (Japanese Patent Laid-open (Kokai) No.2002-233384).

Furthermore, examples of methods for imparting or enhancingL-cysteine-producing ability also include, for example, a method ofenhancing the L-cysteine excretory system, and a method of enhancing thesulfate/thiosulfate transport system. Examples of proteins of theL-cysteine excretory system include the protein encoded by the ydeD gene(Japanese Patent Laid-open (Kokai) No. 2002-233384), the protein encodedby the yfiK gene (Japanese Patent Laid-open (Kokai) No. 2004-49237), theproteins encoded by the emrAB, emrKY, yojIH, acrEF, bcr, and cusA genes(Japanese Patent Laid-open (Kokai) No. 2005-287333), and the proteinencoded by the yeaS gene (Japanese Patent Laid-open (Kokai) No.2010-187552). Examples of the proteins of the sulfate/thiosulfatetransport system include the proteins encoded by the cysPTWAM genecluster.

Specific examples of L-cysteine-producing bacteria and parent strainswhich can be used to derive them include, for example, E. coli JM15transformed with different cysE alleles coding for feedback-resistantserine acetyltransferases (U.S. Pat. No. 6,218,168, Russian patentapplication 2003121601), E. coli W3110 having an over-expressed geneencoding a protein which promotes secretion of a cytotoxic substance(U.S. Pat. No. 5,972,663), E. coli strains having a reduced cysteinedesulfohydrase activity (JP11155571A2), and E. coli W3110 having anincreased activity of a positive transcriptional regulator for cysteineregulon encoded by the cysB gene (WO0127307A1).

Furthermore, examples of coryneform bacteria having L-cysteine-producingability include, for example, coryneform bacteria having serineacetyltransferase desensitized to feedback inhibition by L-cysteinethereby to show enhanced intracellular serine acetyltransferase activity(Japanese Patent Laid-open (Kokai) No. 2002-233384).

<L-Serine-Producing Microorganisms>

Examples of methods for imparting or enhancing L-serine-producingability include, for example, a method of modifying a bacterium so thatthe bacterium has an increased activity or activities of one or morekinds of enzymes such as the L-serine biosynthesis enzymes (JapanesePatent Laid-open (Kokai) No. 11-253187). Examples of such enzymesinclude, but are not particularly limited to, 3-phosphoglyceratedehydrogenase (serA), phosphoserine transaminase (serC), andphosphoserine phosphatase (serB) (Japanese Patent Laid-open (Kokai) No.11-253187). 3-phosphoglycerate dehydrogenase activity can be enhancedby, for example, introducing a mutant serA gene encoding a mutant3-phosphoglycerate dehydrogenase resistant to feedback inhibition byserine. The mutant 3-phosphoglycerate dehydrogenase is disclosed in, forexample, U.S. Pat. No. 6,180,373.

Examples of L-serine-producing bacteria and parent strains which can beused to derive them include, for example, coryneform bacteria havingresistance to azaserine or β-(2-thienyl)-DL-alanine, and deficient inL-serine decomposition ability (Japanese Patent Laid-open (Kokai) No.11-253187). Specific examples of such coryneform bacteria include, forexample, Brevibacterium flavum AJ13324 (FERMP-16128), which hasresistance to azaserine and is deficient in L-serine decompositionability, and Brevibacterium flavum AJ13325 (FERM P-16129), which hasresistance to β-(2-thienyl)-DL-alanine and is deficient in L-serinedecomposition ability (Japanese Patent Laid-open (Kokai) No. 10-248588).

<Isopropyl Alcohol-Producing Microorganisms>

Microorganisms having an ability to produce isopropyl alcohol have beendescribed in detail in W02013/018734A1.

Examples of a method to impart or increase an ability to produceisopropyl alcohol include, for example, a method of modifying amicroorganism to have an increased activity of one or more enzymes suchas isopropyl alcohol biosynthesis enzymes. Examples of such enzymesinclude, but are not particularly limited to, acetoacetatedecarboxylase, isopropyl alcohol dehydrogenase, CoA-transferase, andthiolase (WO2009/008377A1). In particular, the activities of all four ofthese enzymes can be increased.

Moreover, examples of a method to impart or increase an ability toproduce isopropyl alcohol include, for example, a method of modifying amicroorganism to have a reduced activity of GntR (gntR). GntR refers toa transcription factor which negatively regulates the expression of anoperon encoding gluconate metabolic genes. Specifically, the operonencodes gluconate uptake system and gluconate phosphorylase. Forexample, there are two gluconate metabolic systems, GntI and GntII inEscherichia coli, and GntR suppresses the expression of both of them.

Moreover, in addition to the reduced activity of GntR, the microorganismmay be further modified to have an increased or reduced activity of oneor more enzymes such as other enzymes that can affect an ability toproduce isopropyl alcohol when the activity of GntR is reduced. Such anenzyme is also referred to as “auxiliary enzyme”. Examples of theauxiliary enzyme include glucose-6-phosphate isomerase (pgi),glucose-6-phosphate-1-dehydrogenase (Zwf), and phosphogluconatedehydrogenase (Gnd). Altered patterns in these enzyme activities are notparticularly limited as long as an increased production due to a reducedactivity of GntR is retained or increased further.

Examples of the altered pattern in enzymatic activities of the auxiliaryenzymes can include the patterns below:

(1) retained wild-type activities of glucose-6-phosphate isomerase(pgi), glucose-6-phosphate-1-dehydrogenase (Zwf) and phosphogluconatedehydrogenase (Gnd);

(2) reduced activity of glucose-6-phosphate isomerase (pgi) andincreased activity of glucose-6-phosphate-1-dehydrogenase (Zwf);

(3) reduced activity of glucose-6-phosphate isomerase (pgi), andincreased activity of glucose-6-phosphate-1-dehydrogenase (Zwf), andreduced activity of phosphogluconate dehydrogenase (Gnd).

Among these patterns, the enzyme activity pattern of the auxiliaryenzyme group in the above-described (3) is a particular example in termsof the ability to produce isopropyl alcohol.

Glucose-6-phosphate isomerase (pgi) refers to a generic name of anenzyme that corresponds to the enzyme number 5.3.1.9 based on the reportof the Enzyme Committee of the International Union of Biochemistry (I.U. B.), and catalyzes a reaction to produce D-fructose-6-phosphate fromD-glucose-6-phosphate

Glucose-6-phosphate-1-dehydrogenase (Zwf) refers to a generic name of anenzyme that corresponds to the enzyme number 1.1.1.49 based on thereport of the Enzyme Committee of the International Union ofBiochemistry (I. U. B.), and catalyzes a reaction to produceD-glucono-1,5-lactone-6-phosphate from D-glucose-6-phosphate.

Phosphogluconate dehydrogenase (Gnd) refers to a generic name of anenzyme that corresponds to the enzyme number 1.1.1.44 based on thereport of the Enzyme Committee of the International Union ofBiochemistry (I. U. B.), and catalyzes a reaction to produceD-ribulose-5-phosphate and CO₂ from 6-phospho-D-gluconate.

Examples of the glucose-6-phosphate-1-dehydrogenase (Zwf) gene includethe Zwf genes derived from Deinococcus bacteria such as Deinococcusradiophilus, Acetobacter bacteria such as Acetobacter hansenii,Thermotoga bacteria such as Thermotoga maritima, Pseudomonas bacteriasuch as Pseudomonas fluorescens and Pseudomonas aeruginos, Bacillusbacteria such as Bacillus megaterium, Escherichia bacteria such asEscherichia coli, Aspergillus fungi such as Aspergillus niger andAspergillus aculeatus, Cryptococcus fungi such as Cryptococcusneoformans, Dictyostelium fungi such as Dictyostelium discoideum, andSaccharomyces yeasts such as Saccharomyces cerevisiae. Particularexamples include glucose-6-phosphate-1-dehydrogenase (Zwf) gene includethe Zwf genes derived from Deinococcus bacteria, Acetobacter bacteria,Thermotoga bacteria, Pseudomonas bacteria, Bacillus bacteria,Escherichia bacteria, and Aspergillus fungi, and the Zwf gene derivedfrom Escherichia coli is a particular example.

A microorganism having an ability to produce isopropyl alcohol may havebeen modified to have a reduced lactate dehydrogenase activity. Such amodification inhibits the production of lactic acid and allows themicroorganism to produce isopropyl alcohol in an efficient manner evenunder a culture condition in which oxygen supply is limited. The culturecondition in which oxygen supply is limited generally refers to acondition which includes 0.02 vvm to 2.0 vvm (vvm; ventilation volume[mL]/liquid volume [mL]/time [minute]) and a revolution number of 200 to600 rpm when only air is used as a gas.

Examples of a microorganism having an ability to produce isopropylalcohol can include, for example, the Escherichia coli pIPA/B strain andthe pIaaa/B strain described in WO2009/008377, which have increasedactivities of acetoacetate decarboxylase, isopropyl alcoholdehydrogenase, CoA-transferase, and thiolase and can produce isopropylalcohol from plant-derived raw materials; and the like. Moreover,examples of a microorganism having an ability to produce isopropylalcohol also include, for example, the Escherichia coli pIa/B::atoDABstrain described in WO2009/008377, which has increased activities ofCoA-transferase and thiolase due to increased expression of thecorresponding genes on the genome and has increased activities ofisopropyl alcohol dehydrogenase and acetoacetate decarboxylase due tothe introduction of plasmids carrying the corresponding genes. Otherexamples of a microorganism having an ability to produce isopropylalcohol include, for example, the Escherichia coli strains described inWO2009/094485 and WO2009/046929.

<Acetone-Producing Microorganisms>

Acetone is a precursor of isopropyl alcohol in isopropyl alcoholproduction. Accordingly, an ability to produce acetone can be impartedor increased by utilizing a part of the methods to impart or increase anability to produce isopropyl alcohol. For example, an ability to produceacetone can be imparted or increased by modifying a microorganism tohave an increased activity of one or more enzymes such as theabove-indicated isopropyl alcohol biosynthesis enzymes except forisopropyl alcohol dehydrogenase, that is, acetoacetate decarboxylase,CoA-transferase, and thiolase.

<Ethanol-Producing Microorganisms>

Examples of a microorganism having an ability to produce ethanolinclude, for example, Saccharomyces yeasts as well as bacteria belongingto the genera Acinetobacter, Gluconobacter, Zymomonas, Escherichia,Geobacter, Shewanella, Salmonella, Enterobacter, Klebsiella, Bacillus,Clostridium, Corynebacterium, Lactobacillus, Lactococcus, Oenococcus,Streptococcus, and Eubacterium. Moreover, a method for producingrecombinant microorganisms having an ability to produce ethanol is knownin the art of molecular biology (U.S. Pat. Nos. 7,026,152; 6,849,434;6,333,181; 5,821,093; 5,482,846; 5,424,202; 5,028,539; 5,000,000;5,487,989; 5,554,520; and 5,162,516; and WO2003/025117). Moreover,examples of a microorganism having an ability to produce ethanol includea Corynebacterium glutamicum mutant strain in which the lactatedehydrogenase gene (ldhA) has been deleted and the pyruvatedecarboxylase gene (pdc) and the alcohol dehydrogenase gene (adhB) fromZymomonas mobilis have been introduced, and the same mutant strainexcept that the phosphoenolpyruvate carboxylase gene (ppc) has beenfurther deleted (J Mol Microbiol Biotechnol 2004, 8, 243-254). Moreover,examples of a microorganism having an ability to produce ethanol alsoinclude the E. coli strain KO11 in which the pyruvate formate lyase gene(pfl) and the fumarate reductase gene (frd) have been deleted and thepyruvate decarboxylase gene (pdc) and the alcohol dehydrogenase gene(adhB) from Zymomonas mobilis have been introduced (Ann N Y Acad Sci.2008, 1125, 363-372).

<1,3-Propanediol-Producing Microorganisms>

Examples of a microorganism having an ability to produce 1,3-propanediolinclude, for example, bacteria belonging to the genera Escherichia,Klebsiella, Clostridium, and Lactobacillus. The microorganism having anability to produce 1,3-propanediol can have, for example, (a) at leastone gene encoding a glycerol dehydratase reactivation factor, (b) atleast one gene encoding a glycerol dehydratase reactivation factor, and(c) at least one gene encoding a non-specific catalytic activity toconvert 3-hydroxypropionaldehyde into 1,3-propanediol. Moreover, the1,3-propanediol-producing strain of the genus Clostridium can be astrain in which at least one heterogeneous gene encoding an enzymeinvolved in the B-12-independent 1,3-propanediol pathway has beenintroduced. Examples of such a gene include the dhaB1 gene, the dhaB2gene, and the dhaT gene. As a microorganism having an ability to produce1,3-propanediol, for example, microorganisms indicated in JapaneseTranslation of PCT International Application Publication No. 2010-508013are available.

<Organic Acids-Producing Microorganisms>

Examples of microorganisms having an ability to produce acetic acid,3-hydroxybutyric acid, polyhydroxybutyric acid, itaconic acid, citricacid, and/or butyric acid include, for example, microorganisms describedin Enzyme Handbook (Kyoritsu Shuppan Co., Ltd). Examples of amicroorganism having an ability to produce 3-hydroxyisobutyric acidinclude, for example, microorganisms into which a pathway described inWO2009/135074 or WO2008/145737 is introduced. Examples of amicroorganism having an ability to produce 2-hydroxyisobutyric acidinclude, for example, microorganisms into which a pathway described inWO2009/135074 or WO2009/156214 is introduced. Examples of microorganismhaving an ability to produce each of 3-aminoisobutyric acid andmethacrylic acid include, for example, microorganisms into which apathway described in WO2009/135074 is introduced. Examples of amicroorganism having an ability to produce 6-aminocaproic acid include,for example, microorganisms into which a pathway described inWO2012/177721 is introduced.

<Microorganisms Producing Other Objective Substance>

Examples of a microorganism having an ability to produce propyleneinclude, for example, microorganisms into which a pathway described inUS2012-0329119 is introduced. Examples of a microorganism having anability to produce isoprene include, for example, microorganisms intowhich a pathway described in WO2013179722 is introduced. That is,examples of a microorganism having an ability to produce isopreneinclude, for example, microorganisms in which the isoprene synthaseactivity is increased. Such a microorganism having an ability to produceisoprene may be a microorganism in which, for example, the biosynthesispathway for dimethylallyl pyrophosphate (such as mevalonate pathway andmethylerythritol phosphate pathway), which is a substrate of isoprenesynthase, is further enhanced. Examples of a microorganism having anability to produce 1,3-butanediol include, for example, microorganismsinto which a pathway described in WO2012/177619 is introduced. Examplesof a microorganism having an ability to produce 1,4-butanediol include,for example, microorganisms described in Japanese Patent ApplicationPublication No. Sho-62-285779. Examples of microorganisms having anability to produce each of 1-propanol, 1,3-propanediol, and1,2-propanediol include, for example, microorganisms into which apathway described in WO2012/177599 is introduced. Examples of amicroorganism having an ability to produce ethylene glycol include, forexample, microorganisms into which a pathway described in WO2012/177983is introduced. Examples of a microorganism having an ability to produceisobutanol include, for example, microorganisms into which a pathwaydescribed in WO2012/177601 is introduced.

Moreover, a microorganism having an objective substance-producingability may have been modified to have an increased activity ofprotein(s) involved in glycometabolism and/or energy metabolism. Theactivities of these proteins can be increased, for example, byincreasing the expression of genes encoding these proteins.

Examples of the proteins involved in the glycometabolism includeproteins involved in uptake of saccharides and the glycolysis systemenzymes. Examples of genes coding for a protein involved in theglycometabolism include glucose-6-phosphate isomerase gene (pgi,WO01/02542), phosphoenolpyruvate synthase gene (pps, European PatentLaid-open No. 877090), phosphoenolpyruvate carboxylase gene (ppc,WO95/06114), pyruvate carboxylase gene (pyc, WO99/18228, European PatentLaid-open No. 1092776), phosphoglucomutase gene (pgm, WO03/04598),fructose bisphosphate aldolase gene (pfkB, fbp, WO03/04664), pyruvatekinase gene (pykF, WO03/008609), transaldolase gene (talB, WO03/008611),fumarase gene (fum, WO01/02545), non-PTS sucrose uptake gene (csc,European Patent Laid-open No. 149911), and sucrose assimilation gene(scrAB operon, WO90/04636).

Examples of genes encoding the proteins involved in the energymetabolism include the transhydrogenase gene (pntAB, U.S. Pat. No.5,830,716) and cytochrome bo-type oxidase gene (cyoB, European PatentLaid-open No. 1070376).

Moreover, when glycerol is used as a carbon source, for increasing theability to assimilate glycerol, a microorganism having an objectivesubstance-producing ability may have been modified so that theexpression of the glpR gene is attenuated (EP1715056) or the expressionof genes involved in glycerol metabolism, such as the glpA, glpB, glpC,glpD, glpE, glpF, glpG, glpK, glpQ, glpT, glpX, tpiA, gldA, dhaK, dhaL,dhaM, dhaR, fsa and talC genes (EP1715055A), is increased.

Moreover, a microorganism having an objective substance-producingability may have been modified to have an increased activity to exportthe objective substance from cells of the microorganism. The activity toexport an objective substance can be increased, for example, byincreasing the expression of a gene encoding a protein for the export ofthe objective substance. For example, examples of genes encodingproteins for the export of various amino acids include the b2682 geneand the b2683 gene (ygaZH gene) (EP 1239041 A2).

The genes used for the breeding of the aforementioned objectivesubstance-producing microorganisms are not limited to the genesexemplified above and genes having a known nucleotide sequence, and maybe variants thereof, so long as they encode proteins of which theoriginal functions are maintained. For example, the genes used for thebreeding of the objective substance-producing microorganisms may be agene coding for a protein having an amino acid sequence of a knownprotein, but include substitution, deletion, insertion, or addition ofone or several amino acid residues at one or several positions. For thevariants of genes and proteins, the descriptions for variants ofdicarboxylic acid exporter proteins and genes encoding the exporterproteins mentioned later can be applied, mutatis mutandis.

<1-2> Reduction in the Dicarboxylic Acid Exporter Protein Activity

The microorganism of the present invention has been modified so that theactivity of a dicarboxylic acid exporter protein is reduced. Themicroorganism of the present invention can be obtained by modifying amicroorganism having an objective substance-producing ability asdescribed above so that the activity of a dicarboxylic acid exporterprotein is reduced. Moreover, the microorganism of the present inventioncan also be obtained by modifying a microorganism so that the activityof a dicarboxylic acid exporter protein is reduced and subsequentlyimparting or enhancing an objective substance-producing ability.Additionally, the microorganism of the present invention may be amicroorganism which has acquired an objective substance-producingability because of the modification to reduce the dicarboxylic acidexporter protein activity. The modification to establish themicroorganism of the present invention may be performed in an arbitraryorder.

The term “dicarboxylic acid exporter protein” refers to a protein havingan activity to export a dicarboxylic acid having four or five carbonatoms (C₄-C₅ dicarboxylic acid). Examples of a C₄-C₅ dicarboxylic acidinclude, for example, malic acid, fumaric acid, succinic acid,2-hydroxyglutaric acid (also referred to as α-hydroxyglutaric acid), andα-ketoglutaric acid (also referred to as 2-oxoglutaric acid).

The dicarboxylic acid exporter protein activity can be reduced bydisrupting a gene encoding the same protein, and the like. Detailedprocedures to reduce the activity of the protein will be describedbelow. Examples of a gene encoding a dicarboxylic acid exporter proteininclude, for example, the yjjP gene, the yjjB gene, the yeeA gene, theynfM gene, and the sucE1 gene.

<The yjjP Gene>

The yjjP gene is a gene encoding a protein that is predicted to be aninner membrane structural protein. The yjjP gene in the Escherichia colistrain MG1655 is also referred to as b4364 or ECK4354. The nucleotidesequence of the yjjP gene in the Escherichia coli strain MG1655 is shownin SEQ ID NO: 157 and the amino acid sequence of the protein (GenBankAccession No NP_418784.4) encoded by the same gene is shown in SEQ IDNO: 158. Moreover, the nucleotide sequence of the yjjP gene inEnterobacter aerogenes is shown in SEQ ID NO: 159 and the amino acidsequence of the protein encoded by the same gene is shown in SEQ ID NO:160.

<The yjjB Gene>

The yjjB gene is a gene encoding a protein presumed to be a conservedinner membrane protein. The yjjB gene in the Escherichia coli strainMG1655 is also referred to as b3463 or ECK4353. The nucleotide sequenceof the yjjB gene in the Escherichia coli strain MG1655 is shown in SEQID NO: 161 and the amino acid sequence of the protein (GenBank AccessionNo NP_418783.2) encoded by the same gene is shown in SEQ ID NO: 162.Moreover, the nucleotide sequence of the yjjB gene in Enterobacteraerogenes is shown in SEQ ID NO: 163 and the amino acid sequence of theprotein encoded by the same gene is shown in SEQ ID NO: 164.

<The yeeA Gene>

The yeeA gene is a gene encoding a protein presumed to be a conservedinner membrane protein. The yeeA gene in the Escherichia coli strainMG1655 is also referred to as b2008 or ECK2002. The nucleotide sequenceof the yeeA gene in the Escherichia coli strain MG1655 is shown in SEQID NO: 165 and the amino acid sequence of the protein (GenBank AccessionNo NP_416512.1) encoded by the same gene is shown in SEQ ID NO: 166.Moreover, the nucleotide sequence of the yeeA gene in the Pantoeaananatis strain AJ13355 is shown in SEQ ID NO: 167 and the amino acidsequence of the protein encoded by the same gene is shown in SEQ ID NO:168. Moreover, the nucleotide sequence of the yeeA gene in Enterobacteraerogenes is shown in SEQ ID NO: 169 and the amino acid sequence of theprotein encoded by the same gene is shown in SEQ ID NO: 170.

<The ynfM Gene>

The ynfM gene is a gene encoding a protein presumed to be a predictedtransport protein YnfM. The ynfM gene in the Escherichia coli strainMG1655 is also referred to as b1596 or ECK1591. The nucleotide sequenceof the ynfM gene in the Escherichia coli strain MG1655 is shown in SEQID NO: 171 and the amino acid sequence of the protein (GenBank AccessionNo NP_416113.1) encoded by the same gene is shown in SEQ ID NO: 172.Moreover, the nucleotide sequence of the ynfM gene in the Pantoeaananatis strain AJ13355 is shown in SEQ ID NO: 173 and the amino acidsequence of the protein encoded by the same gene is shown in SEQ ID NO:174. Moreover, the nucleotide sequence of the ynfM gene in Enterobacteraerogenes is shown in SEQ ID NO: 175 and the amino acid sequence of theprotein encoded by the same gene is shown in SEQ ID NO: 176. Moreover,the nucleotide sequence of the ynfM gene in Corynebacterium glutamicumATCC13032 is shown in SEQ ID NO: 177 and the amino acid sequence of theprotein (GenBank Accession No NP_602116.1) encoded by the same gene isshown in SEQ ID NO: 178. Moreover, the nucleotide sequence of the ynfMgene in Corynebacterium glutamicum ATCC 13869 is shown in SEQ ID NO: 179and the amino acid sequence of the protein encoded by the same gene isshown in SEQ ID NO: 180.

<The sucE1 Gene>

The sucE1 gene is a gene encoding a protein presumed to be a succinateexporter. The sucE1 gene in Corynebacterium glutamicum ATCC13032 is alsoreferred to as NCgl2130. The nucleotide sequence of the sucE1 gene inCorynebacterium glutamicum ATCC13032 is shown in SEQ ID NO: 277 and theamino acid sequence of the protein (GenBank Accession No NP_601414.1)encoded by the same gene is shown in SEQ ID NO: 278. Moreover, thenucleotide sequence of the sucE1 gene in Corynebacterium glutamicumATCC13869 is shown in SEQ ID NO: 279 and the amino acid sequence of theprotein encoded by the same gene is shown in SEQ ID NO: 280.

Moreover, the activity of a dicarboxylic acid exporter protein newlyidentified by screening may be reduced. The screening for a dicarboxylicacid exporter protein can be performed by, for example, a method asdescribed below. The P. ananatis strain SC17(0) ΔsdhA/RSFPP can be usedas a host for the screening. This strain is a strain in which asuccinate dehydrogenase is deficient and, furthermore, the expression ofthe ppc and prpC genes is increased by introducing the RSFPP plasmid.The RSFPP plasmid is obtained by deleting a region including the gdhAgene from the RSFPPG plasmid (WO2010/027022A1) (see EXAMPLES below).This strain produces succinic acid under aerobic conditions, and thegrowth of this strain is inhibited when succinic acid is present in amedium at a low pH. Utilizing this character enables the screening ofdicarboxylic acid exporter proteins, in which a minimal media at pH 4.7containing 1 to 20 mM succinic acid is used as a medium for thescreening and genomic libraries of E. coli, P. anantis, E. aerogenes,and the like are introduced to this strain to obtain resistant strains.Moreover, the P. ananatis strain SC17(0)ΔsdhAΔyeeA/RSFPP, the P.ananatis strain SC17(0)ΔsdhAΔynfM/RSFPP, or the P. ananatis strainSC17(0)ΔsdhAΔyeeAΔynfM/RSFPP, all of which are deficient in yeeA and/orynJM, can also be used as a host for the screening.

In the present invention, the activity of one type of dicarboxylic acidexporter protein may be reduced, or the activities of two or more typesof dicarboxylic acid exporter proteins may be reduced.

The reduction in the dicarboxylic acid exporter protein activity can beconfirmed by, for example, identifying a reduced ability to producesuccinic acid. Specifically, for example, whether a certain modifiedgene encodes a dicarboxylic acid exporter protein having a loweractivity than that of the wild-type (non-modified) dicarboxylic acidexporter protein can be confirmed by introducing the modified gene intoa strain having a significantly reduced ability to export succinic acidand identifying a reduced ability to produce succinic acid in the strainas compared with a strain in which the non-modified gene has beenintroduced. Examples of a strain having a significantly reduced abilityto export succinic acid include, for example, P. ananatisSC17(0)ΔsdhAΔyeeAΔynfM/RSFPP and C. glutamicum ΔldhΔsucE1 (Fukui et al.,J. Biotechnol, 154(2011)25-34). Moreover, the reduction in thedicarboxylic acid exporter protein activity can also be confirmed by,for example, identifying a decreased amount of the corresponding mRNA orthe corresponding protein.

The dicarboxylic acid exporter protein may be a variant of any of theaforementioned dicarboxylic acid exporter proteins such as proteinsencoded by the various yjjP, yjjB, yeeA, ynJM, and sucE1 genes, so longas it has the activity to export a dicarboxylic acid. Such a variant mayalso be referred to as “conservative variant”. Examples of theconservative variant include, for example, homologues and artificiallymodified variants of the aforementioned dicarboxylic acid exporterproteins such as proteins encoded by the various yjjP, yjjB, yeeA, ynJM,and sucE1 genes.

A gene coding for a homologue of the aforementioned dicarboxylic acidexporter protein can easily be obtained from a public database by, forexample, BLAST search or FASTA search using the nucleotide sequence ofthe aforementioned gene coding for the dicarboxylic acid exporterprotein as a query sequence. Furthermore, a gene coding for a homologueof the aforementioned dicarboxylic acid exporter protein can be obtainedby, for example, PCR using the chromosome of a bacterium or yeast as thetemplate, and oligonucleotides prepared on the basis of a known genesequence thereof as primers.

The gene coding for a conservative variant of the dicarboxylic acidexporter protein may be, for example, such a gene as mentioned below.That is, the gene coding for the dicarboxylic acid exporter protein maybe a gene coding for a protein having the aforementioned amino acidsequence but including substitution, deletion, insertion, or addition ofone or several amino acid residues at one or several positions, so longas it codes for a protein having the activity to export a dicarboxylicacid. In such a case, usually 70% or more, 80% or more, or 90% or more,of the corresponding activity is maintained in the variant protein,relative to the protein before including addition, deletion, insertion,or addition of one or several amino acid residues. Although the numberof “one or several” may differ depending on the positions in thethree-dimensional structure of the protein or the types of amino acidresidues, specifically, it is 1 to 50, 1 to 40, 1 to 30, 1 to 20, 1 to10, or 1 to 5.

The aforementioned substitution, deletion, insertion, or addition of oneor several amino acid residues is a conservative mutation that maintainsnormal function of the protein. Typical examples of the conservativemutation are conservative substitutions. The conservative substitutionis a mutation wherein substitution takes place mutually among Phe, Trp,and Tyr, if the substitution site is an aromatic amino acid; among Leu,Ile, and Val, if it is a hydrophobic amino acid; between Gln and Asn, ifit is a polar amino acid; among Lys, Arg, and His, if it is a basicamino acid; between Asp and Glu, if it is an acidic amino acid; andbetween Ser and Thr, if it is an amino acid having a hydroxyl group.Examples of substitutions considered as conservative substitutionsinclude, specifically, substitution of Ser or Thr for Ala, substitutionof Gln, His, or Lys for Arg, substitution of Glu, Gln, Lys, His, or Aspfor Asn, substitution of Asn, Glu, or Gln for Asp, substitution of Seror Ala for Cys, substitution of Asn, Glu, Lys, His, Asp, or Arg for Gln,substitution of Gly, Asn, Gln, Lys, or Asp for Glu, substitution of Profor Gly, substitution of Asn, Lys, Gln, Arg, or Tyr for His,substitution of Leu, Met, Val, or Phe for Ile, substitution of Ile, Met,Val, or Phe for Leu, substitution of Asn, Glu, Gln, His, or Arg for Lys,substitution of Ile, Leu, Val, or Phe for Met, substitution of Trp, Tyr,Met, Ile, or Leu for Phe, substitution of Thr or Ala for Ser,substitution of Ser or Ala for Thr, substitution of Phe or Tyr for Trp,substitution of His, Phe, or Trp for Tyr, and substitution of Met, Ile,or Leu for Val. Furthermore, such substitution, deletion, insertion,addition, inversion, or the like of amino acid residues as mentionedabove includes a naturally occurring mutation due to an individualdifference, or a difference of species of the organism from which thegene is derived (mutant or variant).

Furthermore, the gene having such a conservative mutation as mentionedabove may be a gene coding for a protein showing a homology of 80% ormore, 90% or more, 95% or more, 97% or more, or 99% or more, to thetotal amino acid sequence mentioned above, and having the activity toexport a dicarboxylic acid. In addition, in this specification,“homology” means “identity”.

Moreover, the gene coding for the dicarboxylic acid exporter protein maybe a DNA that is able to hybridize under stringent conditions with aprobe that can be prepared from a known gene sequence, such as asequence complementary to a part or the whole of the aforementionednucleotide sequence, and which DNA codes for a protein having theactivity to export a dicarboxylic acid. The “stringent conditions” referto conditions under which a so-called specific hybrid is formed, and anon-specific hybrid is not formed. Examples of the stringent conditionsinclude those under which highly homologous DNAs hybridize to eachother, for example, DNAs not less than 80% homologous, not less than 90%homologous, not less than 95% homologous, not less than 97% homologous,or not less than 99% homologous, hybridize to each other, and DNAs lesshomologous than the above do not hybridize to each other, or conditionsof washing of typical Southern hybridization, i.e., conditions ofwashing once, preferably 2 or 3 times, at a salt concentration andtemperature corresponding to 1×SSC, 0.1% SDS at 60° C., 0.1×SSC, 0.1%SDS at 60° C., or 0.1×SSC, 0.1% SDS at 68° C.

The probe used for the aforementioned hybridization may be a part of asequence that is complementary to the gene as described above. Such aprobe can be prepared by PCR using oligonucleotides prepared on thebasis of a known gene sequence as primers and a DNA fragment containingthe nucleotide sequence as a template. As the probe, for example, a DNAfragment having a length of about 300 bp can be used. When a DNAfragment having a length of about 300 bp is used as the probe, thewashing conditions of the hybridization may be, for example, 50° C.,2×SSC and 0.1% SDS.

Furthermore, the gene coding for the dicarboxylic acid exporter proteinmay be a gene in which an arbitrary codon is replaced with an equivalentcodon, so long as the gene codes for a protein having the activity toexport a dicarboxylic acid. For example, the gene coding for thedicarboxylic acid exporter protein may be modified so that it hasoptimal codons according to codon frequencies in a host to be used.

The percentage of the sequence identity between two sequences can bedetermined by, for example, using a mathematical algorithm. Non-limitingexamples of such a mathematical algorithm include the algorithm of Myersand Miller (1988) CABIOS 4:11-17, the local homology algorithm of Smithet al (1981) Adv. Appl. Math. 2:482, the homology alignment algorithm ofNeedleman and Wunsch (1970) J. Mol. Biol. 48:443-453, the method forsearching homology of Pearson and Lipman (1988) Proc. Natl. Acad. Sci.85:2444-2448, and an modified version of the algorithm of Karlin andAltschul (1990) Proc. Natl. Acad. Sci. USA 87:2264, such as thatdescribed in Karlin and Altschul (1993) Proc. Natl. Acad. Sci. USA90:5873-5877.

By using a program based on such a mathematical algorithm, sequencecomparison (i.e. alignment) for determining the sequence identity can beperformed. The program can be appropriately executed by a computer.Examples of such a program include, but are not limited to, CLUSTAL ofPC/Gene program (available from Intelligenetics, Mountain View, Calif.),ALIGN program (Version 2.0), and GAP, BESTFIT, BLAST, FASTA, and TFASTAof Wisconsin Genetics Software Package, Version 8 (available fromGenetics Computer Group (GCG), 575 Science Drive, Madison, Wis., USA).Alignment using these programs can be performed by using, for example,initial parameters. The CLUSTAL program is well described in Higgins etal. (1988) Gene 73:237-244 (1988), Higgins et al. (1989) CABIOS5:151-153, Corpet et al. (1988) Nucleic Acids Res. 16:10881-90, Huang etal. (1992) CABIOS 8:155-65, and Pearson et al. (1994) Meth. Mol. Biol.24:307-331.

In order to obtain a nucleotide sequence homologous to a targetnucleotide sequence, in particular, for example, BLAST nucleotide searchcan be performed by using BLASTN program with score of 100 and wordlength of 12. In order to obtain an amino acid sequence homologous to atarget protein, in particular, for example, BLAST protein search can beperformed by using BLASTX program with score of 50 and word length of 3.See http://www.ncbi.nlm.nih.gov for BLAST nucleotide search and BLASTprotein search. In addition, Gapped BLAST (BLAST 2.0) can be used inorder to obtain an alignment including gap(s) for the purpose ofcomparison. In addition, PSI-BLAST can be used in order to performrepetitive search for detecting distant relationships between sequences.See Altschul et al. (1997) Nucleic Acids Res. 25:3389 for Gapped BLASTand PSI-BLAST. When using BLAST, Gapped BLAST, or PSI-BLAST, initialparameters of each program (e.g. BLASTN for nucleotide sequences, andBLASTX for amino acid sequences) can be used. An alignment can also bemanually performed.

The sequence identity between two sequences is calculated as the ratioof residues matching in the two sequences when aligning the twosequences so as to fit maximally with each other.

The above descriptions concerning conservative variants of genes andproteins can also be applied mutatis mutandis to other proteins such asα-ketoglutarate synthase and genes coding for them.

<1-3> Additional Modifications

The microorganism of the present invention may further have othermodification(s). The other modification(s) can be appropriately selecteddepending on the type of the objective substance, the type of themicroorganism, and the like.

For example, the microorganism of the present invention may have beenmodified to have an attenuated reaction for oxidizing NADH. For example,the microorganism of the present invention may have been modified tohave an attenuated reaction related to energy metabolism and/or anattenuated biosynthetic system for pyruvic acid or acetyl-CoA-derivedsubstances, the reaction and system involving the oxidation of NADH, sothat the reaction of oxidizing NADH is attenuated.

Attenuation of a reaction related to energy metabolism, the reactioninvolving the oxidation of NADH, can be achieved by reducing theactivity of one or more types of enzymes involved in the same reaction.The enzymes involved in the reaction may each be an enzyme directlyoxidizing NADH or an enzyme indirectly oxidizing NADH through couplingwith other enzyme(s) and the like. Examples of enzymes involved in thesame reaction include, for example, NADH dehydrogenase andmalate:quinone oxidoreductase (MQO). NADH dehydrogenase is an enzymewhich directly oxidizes NADH. MQO is an enzyme which indirectly oxidizesNADH. For example, the activity of either NADH dehydrogenase or MQO maybe reduced, or the activities of both enzymes may be reduced.

Attenuation of a biosynthetic system for pyruvic acid oracetyl-CoA-derived substances, the system involving the oxidation ofNADH, can be achieved by reducing the activity of one or more types ofenzymes in the same biosynthetic system. Examples of enzymes in the samebiosynthetic system include, for example, enzymes as indicated below(WO2009/072562):

lactate dehydrogenase (lactate biosynthetic system);

alcohol dehydrogenase (ethanol biosynthetic system);

acetolactate synthase, acetolactate decarboxylase, and acetoin reductase(2,3-butanediol biosynthetic system).

That is, specifically, the microorganism of the present invention mayhave been modified, for example, to have (a) a reduced activity of oneor more types of enzymes such as NADH dehydrogenase and malate:quinoneoxidoreductase, and/or (b) a reduced activity of one or more types ofenzymes such as lactate dehydrogenase, alcohol dehydrogenase,acetolactate synthase, acetolactate decarboxylase, and acetoinreductase. The activity of an enzyme can be reduced by disrupting a geneencoding the enzyme, and the like. Detailed procedures to reduce theactivity of the enzyme will be described below.

The term “NADH dehydrogenase” refers to a protein having an activity tocatalyze a reaction for oxidizing NADH by using a quinone as an electronacceptor. Moreover, this activity is also referred to as “NADHdehydrogenase activity”. NADH dehydrogenases are classified into type Iand type II. The activity of either type I or type II may be reduced, orthe activities of both types may be reduced.

Type I NADH dehydrogenase is a NADH dehydrogenase which has aproton-exporting ability and is also referred to as NDH-1 orNADH:ubiquinone reductase. A type I NADH dehydrogenase specificallycatalyzes the reaction below:

NADH+quinone+5H⁺ _(in)→NAD⁺+quinol+4H⁺ _(out)  (EC 1.6.5.3).

Examples of genes encoding NDH-1 include, the nuoABCEFGHIJKLMN operon(also referred to as “the nuo operon”). The nuo operon encodes subunitsbelow and these subunits form a complex that functions as NDH-1:

Membrane subunit A, H, J, K, L, M, N

membrane subunit A=NuoA (nuoA),

membrane subunit H=NuoH (nuoH),

membrane subunit J=NuoJ (nuoJ),

membrane subunit K=NuoK (nuoK),

membrane subunit L=NuoL (nuoL),

membrane subunit M=NuoM (nuoM),

membrane subunit N=NuoN (nuoN);

soluble NADH dehydrogenase fragment, chain E, F, G

NADH: ubiquinone oxidoreductase, chain E=NuoE (nuoE),

NADH: ubiquinone oxidoreductase, chain F=NuoF (nuoF),

NADH:ubiquinone oxidoreductase, chain G=NuoG (nuoG);

connecting fragment of NADH dehydrogenase I, chain B, CD, I

NADH:ubiquinone oxidoreductase, chain B=NuoB (nuoB),

NADH:ubiquinone oxidoreductase, chain CD=NuoC (nuoC),

NADH: ubiquinone oxidoreductase, chain I=NuoI (nuoI).

The nuoABCEFGHIJKLMN operon in E. coli MG1655 corresponds to a sequencecomplementary to a sequence from position 2388070 to 2403094 of agenomic sequence registered as GenBank accession NC_000913 (VERSIONNC_000913.2 GI:49175990) in the NCBI database. The nucleotide sequenceof the nuoABCEFGHIJKLMN operon in E. coli MG1655 is shown in SEQ IDNO: 1. Moreover, the position of the coding region of each gene(excluding its termination codon) in SEQ ID NO: 1 and the sequenceidentification number for the amino acid sequence of a subunit encodedby each gene are as shown below:

nuoA; 1-441 (NuoA; SEQ ID NO: 2),

nuoB; 460-1119 (NuoB; SEQ ID NO: 3),

nuoC; 1228-3015 (NuoC; SEQ ID NO: 4),

nuoE; 3021-3518 (NuoE; SEQ ID NO: 5),

nuoF; 3518-4852 (NuoF; SEQ ID NO: 6),

nuoG; 4908-7631 (NuoG; SEQ ID NO: 7),

nuoH; 7631-8605 (NuoH; SEQ ID NO: 8),

nuoI; 8623-9162 (NuoI; SEQ ID NO: 9),

nuoJ; 9177-9728 (NuoJ; SEQ ID NO: 10),

nuoK; 9728-10027 (NuoK; SEQ ID NO: 11),

nuoL; 10027-11865 (NuoL; SEQ ID NO: 12),

nuoM; 12032-13558 (NuoM; SEQ ID NO: 13),

nuoN; 13568-15022 (NuoN; SEQ ID NO: 14).

The nuoABCEFGHIJKLMN operon in Pantoea ananatis LMG20103 corresponds toa sequence complementary to a sequence from position 2956133 to 2971292of a genomic sequence registered as GenBank accession NC_013956 (VERSIONNC_013956.2 GI:332139403) in the NCBI database. Moreover, thenuoABCEFGHIJKLMN operon in Pantoea ananatis AJ13355 corresponds to asequence complementary to a sequence from position 2333027 to 2348186 ofa genomic sequence registered as GenBank accession NC_017531 (VERSIONNC_017531.1 GI:386014600) in the NCBI database. The nucleotide sequenceof the nuoABCEFGHIJKLMN operon in Pantoea ananatis AJ13355 is shown inSEQ ID NO: 15. Moreover, the position of the coding region of each nuogene (excluding its termination codon) in SEQ ID NO: 15 and the sequenceidentification number for the amino acid sequence of a subunit encodedby each nuo gene are as shown below:

nuoA; 1-441 (NuoA; SEQ ID NO: 16),

nuoB; 460-1134 (NuoB; SEQ ID NO: 17),

nuoC; 1255-3051 (NuoC; SEQ ID NO: 18),

nuoE; 3057-3569 (NuoE; SEQ ID NO: 19),

nuoF; 3569-4912 (NuoF; SEQ ID NO: 20),

nuoG; 5027-7747 (NuoG; SEQ ID NO: 21),

nuoH; 7747-8721 (NuoH; SEQ ID NO: 22),

nuoI; 8736-9275 (NuoI; SEQ ID NO: 23),

nuoJ; 9238-9837 (NuoJ; SEQ ID NO: 24),

nuoK; 9837-10136 (NuoK; SEQ ID NO: 25),

nuoL; 10136-11968 (NuoL; SEQ ID NO: 26),

nuoM; 12281-13693 (NuoM; SEQ ID NO: 27),

nuoN; 13703-15157 (NuoN; SEQ ID NO: 28).

Incidentally, coryneform bacteria have no NDH-1.

The activity of NDH-1 can be reduced by modifying one or more genesselected from the nuo operon. Moreover, the activity of NDH-1 can alsobe reduced through a reduction of all transcription from the nuo operonby modifying a region that affects the transcription of the nuo operon(e.g. promoter element and/or SD sequence).

Type II NADH dehydrogenase is a NADH dehydrogenase which does not have aproton-exporting ability and is also referred to as NDH-2, NADH dhII, orNADH dehydrogenase II. A type II NADH dehydrogenase specificallycatalyzes the reaction below:

NADH+H⁺+quinone→NAD⁺+quinol  (EC1.6.99.3 or EC 1.6.99.5).

Examples of a gene encoding NDH-2 include the ndh gene.

The ndh gene in E. coli MG1655 corresponds to a sequence from position1165308 to 1166612 of a genomic sequence registered as GenBank accessionNC 000913 (VERSION NC_000913.2 GI:49175990) in the NCBI database. Thendh gene in E. coli MG1655 is synonymous with ECK1095 and JW1095.Moreover, the Ndh protein in E. coli MG1655 is registered as GenBankaccession NP_415627 (version NP_415627.1 GI:16129072,locus_tag=“b1109”). The nucleotide sequence of the ndh gene in E. coliMG1655 and the amino acid sequence of the Ndh protein encoded by thesame gene are shown in SEQ ID NOs: 29 and 30, respectively.

The ndh gene in Pantoea ananatis LMG20103 corresponds to a sequence fromposition 1685397 to 1686704 of a genomic sequence registered as GenBankaccession NC_013956 (VERSION NC_013956.2 GI:332139403) in the NCBIdatabase. Moreover, the Ndh protein in Pantoea ananatis LMG20103 isregistered as GenBank accession YP_003519794 (version YP_003519794.1GI:291617052, locus_tag=“PANA_1499”). Moreover, the ndh gene in Pantoeaananatis AJ13355 corresponds to a sequence from position 1000123 to1001370 of a genomic sequence registered as GenBank accession NC_017531(VERSION NC_017531.1 GI:386014600) in the NCBI database. Moreover, theNdh protein in Pantoea ananatis AJ13355 is registered as GenBankaccession YP_005933721 (version YP_005933721.1 GI:386015440). Thenucleotide sequence of the ndh gene in Pantoea ananatis AJ13355 and theamino acid sequence of the Ndh protein encoded by the same gene areshown in SEQ ID NOs: 31 and 32, respectively.

The ndh gene in Corynebacterium glutamicum ATCC13032 corresponds to asequence complementary to a sequence from position 1543151 to 1544554 ofa genomic sequence registered as GenBank accession NC_003450 (VERSIONNC_003450.3 GI:58036263) in the NCBI database. The ndh gene inCorynebacterium glutamicum ATCC13032 is synonymous with Cgl1465.Moreover, the Ndh protein in Corynebacterium glutamicum ATCC13032 isregistered as GenBank accession NP_600682 (version NP_600682.1GI:19552680, locus_tag=“NCgl1409”). The nucleotide sequence of the ndhgene in Corynebacterium glutamicum ATCC13032 and the amino acid sequenceof the Ndh protein encoded by the same gene are shown in SEQ ID NOs: 85and 86, respectively.

The reduction in the NADH dehydrogenase activity can be confirmed, forexample, by measuring the NADH dehydrogenase activity. The activity ofeither type I NADH dehydrogenase or type II NADH dehydrogenase can bemeasured by a known method (Journal of Biotechnology 158 (2012) p215-223). In the same method, the reduction in NADH concentration isdetermined using a solution of a solubilized membrane fraction bymeasuring the absorbance at 340 nm to calculate the total activity ofthe NADH dehydrogenase (the sum of the type I NADH dehydrogenaseactivity and the type II NADH dehydrogenase activity), and the type IINADH dehydrogenase activity can be calculated indirectly by subtractingthe value of the type I NADH dehydrogenase activity from the value ofthe total activity.

The term “malate:quinone oxidoreductase” refers to a protein having anactivity to catalyze a reaction for oxidizing malic acid by using aquinone as an electron acceptor. Moreover, this activity is alsoreferred to as “malate:quinone oxidoreductase activity”. A malate:quinone oxidoreductase specifically catalyzes the reaction below:

(S)-malate+quinone→oxaloacetate+quinol  (EC 1.1.99.16).

The malate:quinone oxidoreductase is coupled with the NAD-malatedehydrogenase to form a cycle between malate and oxaloacetate andthereby provides net oxidation of NADH.

Examples of a gene encoding a malate:quinone oxidoreductase include themqo gene.

The mqo gene in E. coli MG1655 corresponds to a sequence complementaryto a sequence from position 2303130 to 2304776 of a genomic sequenceregistered as GenBank accession NC_000913 (VERSION NC_000913.2GI:49175990) in the NCBI database. The mqo gene in E. coli MG1655 issynonymous with ECK2202, JW2198, and yojH. Moreover, the Mqo protein inE. coli MG1655 is registered as GenBank accession NP_416714 (versionNP_416714.1 GI:16130147, locus_tag=“b2210”). The nucleotide sequence ofthe mqo gene in E. coli MG1655 and the amino acid sequence of the Mqoprotein encoded by the same gene are shown in SEQ ID NOs: 33 and 34,respectively.

Moreover, for example, some Pantoea bacteria have two copies of malate:quinone oxidoreductase genes (hereinafter also referred to as “the mqo1gene” and “the mqo2 gene”). The mqo1 and mqo2 genes in Pantoea ananatisLMG20103 correspond to a sequence from position 4213429 to 4215042 and asequence complementary to a sequence from position 4560249 to 4561898 ofa genomic sequence registered as GenBank accession NC_013956 (VERSIONNC_013956.2 GI:332139403) in the NCBI database, respectively. Moreover,the proteins encoded by the mqo1 and mqo2 genes in Pantoea ananatisLMG20103 are registered as GenBank accession YP_003522102 (versionYP_003522102.1 GI:291619360, locus_tag=“PANA_3807”) and GenBankaccession YP_003522407 (version YP_003522407.1 GI:291619665,locus_tag=“PANA_4112”), respectively. Moreover, the mqo1 and mqo2 genesin Pantoea ananatis AJ13355 correspond to a sequence from position197167 to 198816 and a sequence from position 3620570 to 3622183 of agenomic sequence registered as GenBank accession NC_017531 (VERSIONNC_017531.1 GI:386014600) in the NCBI database, respectively. Moreover,the proteins encoded by the mqo1 and mqo2 genes in Pantoea ananatisAJ13355 are registered as GenBank accession YP_005941143 (versionYP_005941143.1 GI:386018537) and GenBank accession YP_005935901 (versionYP_005935901.1 GI:386017603), respectively. The nucleotide sequence ofthe mqo1 gene in Pantoea ananatis AJ13355 and the amino acid sequence ofthe protein encoded by the same gene are shown in SEQ ID NOs: 35 and 36,respectively. The nucleotide sequence of the mqo2 gene in Pantoeaananatis AJ13355 and the amino acid sequence of the protein encoded bythe same gene are shown in SEQ ID NOs: 97 and 98, respectively.

The mqo gene in Corynebacterium glutamicum ATCC13032 corresponds to asequence complementary to a sequence from position 2113861 to 2115363 ofa genomic sequence registered as GenBank accession NC_003450 (VERSIONNC_003450.3 GI:58036263) in the NCBI database. The mqo gene inCorynebacterium glutamicum ATCC13032 is synonymous with Cgl2001.Moreover, the Mqo protein in Corynebacterium glutamicum ATCC13032 isregistered as GenBank accession NP_601207 (version NP_601207.1GI:19553205, locus_tag=“NCgl1926”). The nucleotide sequence of the mqogene in Corynebacterium glutamicum ATCC13032 and the amino acid sequenceof the Mqo protein encoded by the same gene are shown in SEQ ID NOs: 87and 88, respectively.

The malate:quinone oxidoreductase activity can be measured by a knownmethod (Eur. J. Biochem. 254 (1998) 395-403).

The term “lactate dehydrogenase” refers to an enzyme which catalyzes areaction for the production of lactic acid from pyruvic acid by usingNADH or NADPH as an electron donor. Moreover, the activity to catalyzethis reaction is also referred to as “lactate dehydrogenase activity”.Lactate dehydrogenases are broadly classified into L-lactatedehydrogenase (L-LDH; EC 1.1.1.27), which produces L-lactic acid, andD-lactate dehydrogenase (D-LDH; EC1.1.1.28), which produces D-lacticacid, and either activity may be reduced. The lactate dehydrogenase(LDH) activity can be reduced, for example, by disrupting a geneencoding a lactate dehydrogenase (LDH gene), as described below, and thelike. The nucleotide sequence of the D-LDH gene (ldhA) in Escherichiacoli is shown in SEQ ID NO: 37 and the amino acid sequence encoded bythe same gene is shown in SEQ ID NO: 38. The nucleotide sequence of theD-LDH gene (ldhA) in Pantoea ananatis is shown in SEQ ID NO: 39 and theamino acid sequence encoded by the same gene is shown in SEQ ID NO: 40.The nucleotide sequence of the L-LDH gene (ldh) in Corynebacteriumglutamicum ATCC13032 is shown in SEQ ID NO: 229 and the amino acidsequence encoded by the same gene is shown in SEQ ID NO: 230. Thenucleotide sequence of the L-LDH gene (ldh) in the Corynebacteriumglutamicum strain 2256 (ATCC 13869) is shown in SEQ ID NO: 231 and theamino acid sequence encoded by the same gene is shown in SEQ ID NO: 232.The reduction in the lactate dehydrogenase activity can be confirmed,for example, by measuring the lactate dehydrogenase activity with aknown method (L. Kanarek and R. L. Hill, J. Biol. Chem. 239, 4202(1964)). Examples of a specific method to construct a mutant strain ofenteric bacteria having a reduced lactate dehydrogenase activity includea method described in Alam, K. Y, Clark, D. P. 1989. J. Bacteriol. 171:6213-6217, and the like.

The term “alcohol dehydrogenase” refers to an enzyme which catalyzes areaction for the production of alcohol from aldehydes by using NADH orNADPH as an electron donor (EC 1.1.1.1, EC 1.1.1.2, or EC 1.1.1.71).Moreover, the activity to catalyze the same reaction is also referred toas “alcohol dehydrogenase activity”. The alcohol dehydrogenase (ADH)activity can be reduced, for example, by disrupting a gene encoding analcohol dehydrogenase (ADH gene), as described below, and the like. Thenucleotide sequence of the adhE gene as an ADH gene in Escherichia coliis shown in SEQ ID NO: 41 and the amino acid sequence encoded by thesame gene is shown in SEQ ID NO: 42. The nucleotide sequence of the adhEgene as an ADH gene in Pantoea ananatis is shown in SEQ ID NO: 43 andthe amino acid sequence encoded by the same gene is shown in SEQ ID NO:44. The nucleotide sequence of the adhE gene in Corynebacteriumglutamicum ATCC13032 as an ADH gene in Corynebacterium glutamicum isshown in SEQ ID NO: 233 and the amino acid sequence encoded by the samegene is shown in SEQ ID NO: 234. The reduction in the alcoholdehydrogenase activity can be confirmed, for example, by measuring thealcohol dehydrogenase activity with a known method (Lutstorf, U. M.,Schurch, P. M. & von Wartburg, J. P., Eur. J. Biochem. 17, 497-508(1970)). Examples of a specific method to construct a mutant strain ofenteric bacteria having a reduced alcohol dehydrogenase activity includea method described in Sanchez, A. M., Bennett, G. N., San, K.-Y,Biotechnol. Prog. 21, 358-365 (2005), and the like.

The term “acetolactate synthase” refers to an enzyme which catalyzes areaction for the production of an acetolactic acid molecule and CO₂ fromtwo pyruvic acid molecules (EC 2.2.1.6). Moreover, the activity tocatalyze the same reaction is also referred to as “acetolactate synthaseactivity”. In the acetolactate synthase (AHAS), AHAS isozymes I to IIIare known and the activity of any of the isozymes may be reduced. Theacetolactate synthase activity can be reduced, for example, bydisrupting a gene encoding an acetolactate synthase, as described below,and the like. Examples of a gene encoding an acetolactate synthaseinclude the ilvB gene, the ilvG gene, and the ilvI gene, which encodecatalytic subunits of the AHAS I, AHAS II, and AHAS III, respectively.The nucleotide sequences of the ilvB and ilvI genes in E. coli MG1655are shown in SEQ ID NOs: 235 and 237, respectively; and the amino acidsequences of the proteins encoded by the same genes are shown in SEQ IDNOs: 236 and 238, respectively. The nucleotide sequences of the ilvG andilvI genes in Pantoea ananatis AJ13355 are shown in SEQ ID NOs: 239 and241, respectively; and the amino acid sequences of the proteins encodedby the same genes are shown in SEQ ID NOs: 240 and 242, respectively.The nucleotide sequence of the ilvB gene in Corynebacterium glutamicumATCC 13032 is shown in SEQ ID NO: 243 and the amino acid sequence of theprotein encoded by the same gene is shown in SEQ ID NO: 244. Thereduction in the acetolactate synthase activity can be confirmed, forexample, by measuring the acetolactate synthase activity with a knownmethod (F. C. Stormer and H. E. Umbarger, Biochem. Biophys. Res.Commun., 17, 5, 587-592 (1964)).

The term “acetolactate decarboxylase” refers to an enzyme whichcatalyzes a reaction for the production of acetoin by decarboxylation ofacetolactate (EC 4.1.1.5). Moreover, the activity to catalyze the samereaction is also referred to as “the acetolactate decarboxylaseactivity”. The acetolactate decarboxylase activity can be reduced, forexample, by disrupting a gene encoding an acetolactate decarboxylase, asdescribed below, and the like. The nucleotide sequence of theacetolactate decarboxylase gene (budA) in Pantoea ananatis AJ13355 isshown in SEQ ID NO: 245 and the amino acid sequence of the proteinencoded by the same gene is shown in SEQ ID NO: 246. Incidentally, E.coli and Corynebacterium glutamicum have no acetolactate decarboxylase.The reduction in the acetolactate decarboxylase activity can beconfirmed, for example, by measuring the acetolactate decarboxylaseactivity with a known method (Juni E., J. Biol. Chem., 195(2): 715-726(1952)).

The term “acetoin reductase” refers to an enzyme which catalyzes areaction for the production of 2,3-butanediol from acetoin by using NADHor NADPH as an electron donor (EC 1.1.1.4). Moreover, the activity tocatalyze the same reaction is also referred to as “acetoin reductaseactivity”. The acetoin reductase activity can be reduced, for example,by disrupting a gene encoding an acetoin reductase, as described below,and the like. The nucleotide sequence of the acetoin reductase gene(budC) in Pantoea ananatis AJ13355 is shown in SEQ ID NO: 247 and theamino acid sequence of the protein encoded by the same gene is shown inSEQ ID NO: 248. The nucleotide sequence of the acetoin reductase gene(butA) in Corynebacterium glutamicum ATCC 13032 is shown in SEQ ID NO:249 and the amino acid sequence of the protein encoded by the same geneis shown in SEQ ID NO: 250. Incidentally, for example, E. coli has noacetoin reductase. The reduction in the acetoin reductase activity canbe confirmed, for example, by measuring the acetoin reductase activitywith a known method (K. Blomqvist et al., J Bacteriol., 175, 5,1392-1404 (1993)).

Moreover, the microorganism of the present invention may have beenmodified to have an attenuated acetate biosynthetic system.Specifically, the microorganism of the present invention may have beenmodified, for example, to have a reduced activity of one or more enzymesselected from the enzymes below (US2007-0054387, WO2005/052135,WO99/53035, WO2006/031424, WO2005/113745, and WO2005/113744):

phosphotransacetylase;

acetate kinase;

pyruvate oxidase;

acetyl-CoA hydrolase.

The phosphotransacetylase (PTA) activity can be reduced, for example, bydisrupting a gene encoding a phosphotransacetylase (PTA gene), asdescribed below, and the like. The nucleotide sequence of the pta genein Escherichia coli is shown in SEQ ID NO: 45 and the amino acidsequence encoded by the same gene is shown in SEQ ID NO: 46. Thenucleotide sequence of the pta gene in Pantoea ananatis is shown in SEQID NO: 47 and the amino acid sequence encoded by the same gene is shownin SEQ ID NO: 48. The reduction in the phosphotransacetylase activitycan be confirmed by measuring the phosphotransacetylase activity with aknown method (Klotzsch, H. R., Meth. Enzymol. 12, 381-386 (1969)).

Moreover, the microorganism of the present invention may have beenmodified to have a reduced pyruvate formate lyase (PFL) activity. Thepyruvate formate lyase activity can be reduced, for example, bydisrupting a gene encoding a pyruvate formate lyase (PFL gene), asdescribed below, and the like. The nucleotide sequences of the pflB,pflD, and tdcE genes as PFL genes in Escherichia coli are shown in SEQID NOs: 89, 91, and 93, respectively; and the amino acid sequences ofthe proteins encoded by the same genes are shown in SEQ ID NOs: 90, 92,and 94, respectively. The nucleotide sequence of the pflB gene inPantoea ananatis is shown in SEQ ID NO: 95 and the amino acid sequenceof the protein encoded by the same gene is shown in SEQ ID NO: 96. Thereduction in the pyruvate formate lyase activity can be confirmed bymeasuring the pyruvate formate lyase activity with a known method(Knappe, J. & Blaschkowski, H. P., Meth. Enzymol. 41, 508-518 (1975)).

Moreover, the microorganism of the present invention may have beenmodified to have a reduced succinate dehydrogenase (SDH) activity. Thesuccinate dehydrogenase activity can be reduced, for example, bydisrupting a gene encoding a succinate dehydrogenase (SDH gene), asdescribed below, and the like. The reduction in the succinatedehydrogenase activity can be confirmed by measuring the succinatedehydrogenase activity with a known method (Tatsuki Kurokawa and JunshiSakamoto, Arch. Microbiol. 183: 317-324 (2005)).

Moreover, the microorganism of the present invention may have beenmodified to have a reduced activity of a fumarate reductase usingreduced quinone as an electron donor (quinone-fumarate reductase; EC1.3.5.1 or EC 1.3.5.4) and an increased activity of a fumarate reductaseusing NADH as an electron donor (NADH-fumarate reductase; EC 1.3.1.6).Specifically, for example, a gene encoding a quinone-fumarate reductasecan be replaced with a gene encoding a NADH-fumarate reductase. The genereplacement can be performed, for example, by the procedures describedbelow. Examples of a NADH-fumarate reductase include, for example, FRDS1and FRDS2 of Saccharomyces cerevisiae. In Saccharomyces cerevisiae,FRDS1 and FRDS2 are encoded by the FRDS gene and the OSM1 gene,respectively.

Moreover, the microorganism of the present invention may have beenmodified to have an enhanced anaplerotic pathway for the TCA cycle.Specifically, the microorganism of the present invention may have beenmodified, for example, to have an increased activity of one or moreenzymes selected from the enzymes below (Japanese Patent ApplicationPublication No. Hei-11-196888, Japanese Patent Application PublicationNo. 2006-320208, WO99/53035, WO2005/021770; Hong S H, Lee S Y.Biotechnol Bioeng. 74(2): 89-95 (2001); Millard, C. S., Chao, Y P.,Liao, J. C., Donnelly, M. I. Appl. Environ. Microbiol. 62: 1808-1810(1996); Pil Kim, Maris Laivenieks, Claire Vieille, and J. GregoryZeikus. Appl. Environ. Microbiol. 70: 1238-1241 (2004)):

pyruvate carboxylase;

phosphoenolpyruvate carboxylase;

phosphoenolpyruvate carboxykinase.

The activity of an enzyme can be increased, for example, by increasingthe expression of a gene encoding the enzyme, as described below.Examples of a gene encoding a pyruvate carboxylase include, for example,the PC genes of coryneform bacteria such as Corynebacterium glutamicumand Brevibacterium flavum; Bacillus stearothermophilus; Rhizobium etli;and yeasts such as Saccharomyces cerevisiae and Schizosaccharomycespombe (WO2009/072562). Examples of a gene encoding a phosphoenolpyruvatecarboxykinase include, for example, the pckA gene of Actinobacillussuccinogenes (GenBank Accession No. YP_001343536.1), the pckA gene ofHaemophilus influenzae (GenBank Accession No. YP_248516.1), the pckAgene of Pasteurella multocida (GenBank Accession No. NP_246481.1), thepckA gene of Mannheimia succiniciproducens (GenBank Accession No.YP_089485.1), the pckA gene of Yersinia pseudotuberculosis (GenBankAccession No. YP_072243), the pckA gene of Vibrio cholerae (GenBankAccession No. ZP_01981004.1), and the pckA gene of Selenomonasruminantium (GenBank Accession No. AB016600) (WO2009/072562). Examplesof a gene encoding a phosphoenolpyruvate carboxylase include, forexample, the ppc genes of coryneform bacteria such as Corynebacteriumglutamicum and Brevibacterium flavum; Escherichia bacteria such asEscherichia coli; and Rhodopseudomonas palustris. Moreover, the activityof an enzyme can also be increased, for example, by reducing or removingthe feedback inhibition. For example, the phosphoenolpyruvatecarboxylase (PEPC) activity is inhibited by L-malic acid, which is anintermediate of the succinate biosynthesis pathway (Masato Yano andKatsura Izui, Eur. Biochem. FEBS, 247, 74-81, 1997). The inhibition byL-malic acid can be reduced, for example, by introducing adesensitization mutation into PEPC with a single amino acidsubstitution. Specific examples of a desensitization mutation with asingle amino acid substitution include, for example, a mutation causinga substitution of the amino acid at position 620 from lysine to serinein the PEPC protein from Escherichia coli (supra).

Moreover, the microorganism of the present invention may have beenmodified to have an increased α-ketoglutarate synthase (α-KGS) activity.The term “α-ketoglutarate synthase” refers to an enzyme which catalyzesa reaction for the production of α-ketoglutaric acid (2-oxoglutaricacid) from succinyl-CoA and CO₂ bp using reduced ferredoxin or reducedflavodoxin as an electron donor (EC 1.2.7.3). Moreover, the activity tocatalyze the same reaction is also referred to as “α-ketoglutaratesynthase activity”. α-Ketoglutarate synthase is also referred to asα-ketoglutarate oxidoreductase, α-ketoglutarate ferredoxinoxidoreductase, 2-oxoglutarate synthase, 2-oxoglutarate oxidoreductase,or 2-oxoglutarate ferredoxin oxidoreductase. The α-ketoglutaratesynthase is known to function in a multisubunit complex, usually as aheterodimer composed of an α-subunit and a β-subunit. Theα-ketoglutarate synthase activity can be increased, for example, byincreasing the expression of gene(s) encoding an α-ketoglutaratesynthase (α-ketoglutarate synthase gene(s)), as described below.

Examples of α-ketoglutarate synthase genes include, for example, theα-ketoglutarate synthase genes of bacteria in the genera Chlorobium,Desulfobacter, Aquifex, Hydrogenobacter, Thermoproteus, and Pyrobaculum,all of which bacteria possess the reductive TCA cycle, and specificallyinclude the α-ketoglutarate synthase genes of Chlorobium tepidum andHydrogenobacter thermophilus. Moreover, examples of α-ketoglutaratesynthase genes include, for example, the α-ketoglutarate synthase genesof Blastopirellula marina, which is a marine bacterium belonging to theorder Planctomycetes (Schlesner, H. et al. 2004. Int. J. Syst. Evol.Microbiol. 54: 1567-1580), Sulfurimonas denitrificans, which is asulfur-oxidizing bacterium belonging to ε-Proteobacteria (Brinkhoff, T.et al. 1999. Int. J. Syst. Bacteriol. 49:875-879), and Methanococcusmaripaludis, which is a methane-producing bacterium belonging to archaea(Jones, W. J. et al. Arch. Microbiol. 1983. 135: 91-97).

The genomic sequence of Chlorobium tepidum (GenBank Accession No.NC_002932) has been determined (Eisen, J. A. et al. 2002. Proc. Natl.Acad. Sci. USA 99: 9509-9514). The nucleotide sequence of the α-subunitgene of the α-ketoglutarate synthase, which is located in the regionfrom base position 170164 to 172047 (complementary strand) in thegenomic sequence of Chlorobium tepidum, is shown in SEQ ID NO: 49 andthe nucleotide sequence of the β-subunit gene of the same, which islocated in the region from base position 169132 to 170160 (complementarystrand) in the genomic sequence of Chlorobium tepidum, is shown in SEQID NO: 51. Moreover, the amino acid sequence of the α-subunit of theα-ketoglutarate synthase encoded by the same gene (GenBank Accession No.NP_661069) is shown in SEQ ID NO: 50 and the amino acid sequence of theβ-subunit of the same (GenBank Accession No. NP_661068) is shown in SEQID NO: 52. The genomic sequence of Blastopirellula marina (GenBankAccession No. AANZ00000000) has been determined (Fuchsman, C. A., andRocap, G Appl. Environ. Microbiol. 2006. 72: 6841-6844). The nucleotidesequence of the α-subunit gene of the α-ketoglutarate synthase, which islocated in the region from base position 3180 to 5045 (complementarystrand) in the genomic sequence of Blastopirellula marina, is shown inSEQ ID NO: 53 and the nucleotide sequence of the β-subunit gene of thesame, which is located in the region from base position 2089 to 3108(complementary strand) in the genomic sequence of Blastopirellulamarina, is shown in SEQ ID NO: 55. Moreover, the amino acid sequence ofthe α-subunit of the α-ketoglutarate synthase encoded by the same geneis shown in SEQ ID NO: 54 and the amino acid sequence of the β-subunitof the same is shown in SEQ ID NO: 56. Moreover, the α-ketoglutaratesynthase genes of Hydrogenobacter thermophilus (GenBank Accession No.AB046568) have been cloned (Yun, N. R. et al. 2001. Biochem. Biophy.Res. Commum. 282: 589-594) and the α-subunit (GenBank Accession No.BAB21494) and the β-subunit (GenBank Accession No. BAB21495) have beenidentified. Moreover, examples of α-ketoglutarate synthase genesinclude, for example, four genes located in the region from baseposition 620219 to 623070 in the genomic sequence of Helicobacter pylori(GenBank Accession No. NC_00091): HP0588, HP0589, HP0590, and HP0591;and two genes located in the region from base position 2575303 to2578105 in the genomic sequence of Sulfolobus solfataricus (GenBankAccession No. NC 002754): SS02815 and SS02816.

Based on the homology with the above-described genes, α-ketoglutaratesynthase genes cloned from bacteria in the genera Chlorobium,Desulfobacter, , Aquifex, Hydrogenobacter, Thermoproteus, Pyrobaculum,Sulfurimonas, Methanococcus, and the like may also be used as anα-ketoglutarate synthase gene.

An α-subunit gene and β-subunit gene derived from the same organism forthe α-ketoglutarate synthase can be used, but the genes may beindividually derived from different organisms as long as a proteinhaving the α-ketoglutarate synthase activity can be constructed.Examples of a particular combination include, but are not limited to, acombination of an α-subunit having the amino acid sequence of SEQ ID NO:50 or a conservative variant thereof and a β-subunit having the aminoacid sequence of SEQ ID NO: 52 or a conservative variant thereof, and acombination of an α-subunit having the amino acid sequence of SEQ ID NO:54 or a conservative variant thereof and a β-subunit having the aminoacid sequence of SEQ ID NO: 56 or a conservative variant thereof.

The increase in the α-ketoglutarate synthase activity can be confirmed,for example, by preparing crude enzyme solutions from microorganismsbefore and after the modification and comparing the α-ketoglutaratesynthase activity in the crude enzyme solutions. The α-ketoglutaratesynthase activity can be measured according to, for example, a method ofYun et al., (Yun, N. R. et al. 2001. Biochem. Biophy. Res. Commum. 282:589-594). Specifically, the α-ketoglutarate synthase activity can bedetermined by adding α-ketoglutaric acid to a reaction liquid containingoxidized methyl viologen as an electron acceptor, CoA, and a crudeenzyme solution and measuring spectroscopically the amount of reducedmethyl viologen increasing due to the decarboxylation reaction ofα-ketoglutaric acid. One unit (U) of the α-ketoglutarate synthaseactivity is defined as the activity for reducing 1 μmol of methylviologen per one minute. The α-ketoglutarate synthase activity should beincreased at least as compared to that of a non-modified strain. When anon-modified strain has the α-ketoglutarate synthase activity, theα-ketoglutarate synthase activity may be increased preferably 1.5 timesor more, more preferably two times or more, and Furthermore preferablythree times or more as compared to that of the non-modified strain.Moreover, when a non-modified strain does not have the α-ketoglutaratesynthase activity, an α-ketoglutarate synthase should be produced byintroducing α-ketoglutarate synthase genes, and the α-ketoglutaratesynthase may be produced to such a degree that the enzymatic activitycan be determined, or may be produced in an amount of preferably 0.001U/mg or more, more preferably 0.005 U/mg or more, and Furthermorepreferably 0.01 U/mg or more relative to the total protein in microbialcells.

Moreover, the microorganism of the present invention may have beenmodified to have an increased activity to regenerate the reduced form ofan electron donor from the oxidized form of the electron donor, thereduced form being required for the α-ketoglutarate synthase activity.Such a modification may allow the α-ketoglutarate synthase activity tobe increased. Examples of the activity to regenerate the reduced form ofan electron donor from the oxidized form of the electron donor includethe ferredoxin-NADP⁺ reductase activity and the pyruvate synthaseactivity. The activity to regenerate the reduced form of an electrondonor from the oxidized form of the electron donor can be increased, forexample, by increasing the expression of a gene encoding a proteinhaving such an activity. The increase in the activity to regenerate thereduced form of an electron donor from the oxidized form of the electrondonor may or may not be combined with other modifications such as theincrease in the expression of α-ketoglutarate synthase genes.

The term “ferredoxin-NADP⁺ reductase” refers to an enzyme whichreversibly catalyzes the reaction below (EC 1.18.1.2):

reduced ferredoxin+NADP⁺→oxidized ferredoxin+NADPH+H⁺.

In this reaction, ferredoxin can be substituted with flavodoxin. Thatis, ferredoxin-NADP⁺ reductase is synonymous with flavodoxin-NADP⁺reductase. Moreover, ferredoxin-NADP⁺ reductase is also referred to asferredoxin-NADP⁺ oxidoreductase, or NADPH-ferredoxin oxidoreductase. Theabove-described reaction is reversible and a ferredoxin-NADP⁺ reductasecan regenerate the reduced ferredoxin and/or reduced flavodoxin from theoxidized form thereof in the presence of NADPH. That is, by using acombination of a ferredoxin-NADP⁺ reductase and an α-ketoglutaratesynthase, the reduced ferredoxin and/or reduced flavodoxin which hasbeen consumed by the α-ketoglutarate synthase can be regenerated via thereverse reaction of the ferredoxin-NADP⁺ reductase. The ferredoxin-NADP⁺reductase activity can be increased, for example, by increasing the copynumber or expression level of a gene encoding a ferredoxin-NADP⁺reductase (ferredoxin-NADP⁺ reductase gene).

The presence of a ferredoxin-NADP⁺ reductase has been broadly identifiedfrom microorganisms to higher organisms (Carrillo, N. and Ceccarelli, E.A., Eur. J. Biochem. 270: 1900-1915 (2003); Ceccarelli, E. A., et al.,Biochim. Biophys. Acta. 1698: 155-165 (2004)). Examples of aferredoxin-NADP⁺ reductase gene include, for example, the fpr gene inEscherichia coli (Bianchi, V. et al. 1993. J. Bacteriol. 175:1590-1595),the ferredoxin-NADP⁺ reductase gene in Corynebacterium glutamicum, andthe NADPH-putidaredoxin reductase gene in Pseudomonas putida (Koga, H.et al. 1989. J. Biochem. (Tokyo) 106: 831-836).

Specific examples of a flavodoxin-NADP⁺ reductase gene in Escherichiacoli include the fpr gene having the nucleotide sequence of SEQ ID NO:57 and located in the region from base position 4111749 to U.S. Pat. No.4,112,495 (complementary strand) in the genomic sequence of theEscherichia coli strain K-12 (GenBank Accession No. U00096). The aminoacid sequence of the Fpr protein (GenBank Accession No. AAC76906)encoded by the same gene is shown in SEQ ID NO: 58. Moreover, theferredoxin-NADP⁺ reductase gene in Corynebacterium glutamicum (GenBankAccession No. BAB99777) has been identified in the region from baseposition 2526234 to 2527211 in the genomic sequence of Corynebacteriumglutamicum (GenBank Accession No. BA00036).

The increase in the ferredoxin-NADP⁺ reductase activity can beconfirmed, for example, by preparing crude enzyme solutions frommicroorganisms before and after the modification and comparing theferredoxin-NADP⁺ reductase activity in the crude enzyme solutions. Theferredoxin-NADP⁺ reductase activity can be measured according to, forexample, a method of Blaschkowski et al., (Blaschkowski, H. P. et al.1982. Eur. J. Biochem. 123: 563-569). Specifically, the ferredoxin-NADP⁺reductase activity can be determined by using ferredoxin as a substrateand measuring spectroscopically the amount of decreasing NADPH. One unit(U) of the ferredoxin-NADP⁺ reductase activity is defined as theactivity for oxidizing 1 μmol of NADPH per one minute.

The term “pyruvate synthase” refers to an enzyme which reversiblycatalyzes the reaction below for the production of pyruvic acid fromacetyl-CoA and CO₂ by using reduced ferredoxin or reduced flavodoxin asan electron donor (EC 1.2.7.1):

reduced ferredoxin+acetyl-CoA+CO₂→oxidized ferredoxin+pyruvate.

Pyruvate synthase is also referred to as pyruvate oxidoreductase,pyruvate ferredoxin reductase, pyruvate flavodoxin reductase, orpyruvate ferredoxin oxidoreductase. The above-described reaction isreversible and a pyruvate synthase can regenerate the reduced ferredoxinand/or reduced flavodoxin from the oxidized form thereof in the presenceof pyruvic acid. That is, by using a combination of a pyruvate synthaseand an α-ketoglutarate synthase, the reduced ferredoxin and/or reducedflavodoxin which has been consumed by the α-ketoglutarate synthase canbe regenerated via the reverse reaction of the pyruvate synthase. Thepyruvate synthase activity can be increased, for example, by increasingthe copy number or expression level of a gene encoding a pyruvatesynthase (pyruvate synthase gene).

Examples of a pyruvate synthase gene include the pyruvate synthase genesin bacteria possessing the reductive TCA cycle such as Chlorobiumtepidum and Hydrogenobacter thermophilus; the pyruvate synthase genes inbacteria belonging to the family Enterobacteriaceae such as Escherichiacoli; and the pyruvate synthase genes in autotrophic methanogens such asMethanococcus maripaludis, Methanocaldococcus jannaschii, andMethanothermobacter thermautotrophicus.

Specific examples of a pyruvate synthase gene in Chlorobium tepiduminclude a gene having the nucleotide sequence of SEQ ID NO: 59 andlocated in the region from base position 1534432 to 1537989 in thegenomic sequence of Chlorobium tepidum (GenBank Accession No.NC_002932). The amino acid sequence of the pyruvate synthase (GenBankAccession No. AAC76906) encoded by the same gene is shown in SEQ ID NO:60. Moreover, the pyruvate synthase of Hydrogenobacter thermophilus isknown to form a four-subunit complex composed of the δ subunit (GenBankAccession No. BAA95604), the α-subunit (GenBank Accession No. BAA95605),the β-subunit (GenBank Accession No. BAA95606), and the γ subunit(GenBank Accession No. BAA95607) (Ikeda, T. et al. 2006. Biochem.Biophys. Res. Commun. 340: 76-82). Moreover, examples of a pyruvatesynthase gene include, for example, four genes located in the regionfrom base position 1170138 to 1173296 in the genomic sequence ofHelicobacter pylori (GenBank Accession No. NC 000915): HP1108, HP1109,HP1110, and HP1111; and four genes represented by the region from baseposition 1047593 to 1044711 in the genomic sequence of Sulfolobussolfataricus (GenBank Accession No. NC 002754): SS01208, SS07412,SS01207, and SS01206.

Based on the homology with the above-described genes, pyruvate synthasegenes cloned from bacteria in the genera Chlorobium, Desulfobacter,Aquifex, Hydrogenobacter, Thermoproteus, Pyrobaculum and the like may beused as a pyruvate synthase gene.

The increase in the pyruvate synthase activity can be confirmed bypreparing crude enzyme solutions from microorganisms before and afterthe increase and comparing the pyruvate synthase activity in the crudeenzyme solutions. The pyruvate synthase activity can be measuredaccording to, for example, a method of Yoon et al., (Yoon, K. S. et al.1997. Arch. Microbiol. 167: 275-279). The measurement principle is thesame as that for the above-described measurement of the α-ketoglutaratesynthase activity. Specifically, the pyruvate synthase activity can bedetermined by adding pyruvic acid to a reaction liquid containingoxidized methyl viologen as an electron acceptor, CoA, and a crudeenzyme solution and measuring spectroscopically the amount of reducedmethyl viologen increasing due to the decarboxylation reaction of thepyruvic acid. One unit (U) of the pyruvate synthase activity is definedas the activity for reducing 1 μmol of methyl viologen per one minute.The pyruvate synthase activity should be increased at least as comparedto that of a non-modified strain. When a non-modified strain has thepyruvate synthase activity, the pyruvate synthase activity may beincreased preferably 1.5 times or more, more preferably two times ormore, and Furthermore preferably three times or more as compared to thatof the non-modified strain. Moreover, when a non-modified strain doesnot have the pyruvate synthase activity, a pyruvate synthase should beproduced by introducing a pyruvate synthase gene, and the pyruvatesynthase may be produced to such a degree that the enzymatic activitycan be determined, or may be produced in an amount of preferably 0.001U/mg or more, more preferably 0.005 U/mg or more, and Furthermorepreferably 0.01 U/mg or more relative to the total protein in microbialcells.

Moreover, the microorganism of the present invention may have beenmodified to have an increased ability to produce electron donor(s)required for the α-ketoglutarate synthase activity, that is, ferredoxinand/or flavodoxin. Such a modification may allow the α-ketoglutaratesynthase activity to be increased. The ability to produce ferredoxinand/or flavodoxin can be increased, for example, by increasing theexpression of a gene encoding a ferredoxin and/or flavodoxin. Theincrease of the ability to produce ferredoxin and/or flavodoxin may ormay not be combined with other modifications such as the increase in theexpression of α-ketoglutarate synthase genes.

The term “ferredoxin” refers to a protein which functions as aone-electron carrier containing an iron-sulfur cluster. The term“iron-sulfur cluster” refers to a cluster containing non-heme iron atomsand sulfur atoms and is referred to as 4Fe-4S, 3Fe-4S, or 2Fe-2S clusteraccording to the structure of the cluster. The term “flavodoxin” refersto a protein which functions as a one-electron or two-electron carriercontaining FMN (flavin mononucleotide) as a prosthetic group. Ferredoxinand flavodoxin are described in the literature by McLean et al.,(McLean, K. J. et al. 2005. Biochem. Soc. Trans. 33: 796-801).

Genes encoding ferredoxins (ferredoxin genes) and genes encodingflavodoxins (flavodoxin genes) are widely distributed in nature. Anyferredoxin gene or flavodoxin gene may be used as long as it encodes aferredoxin or flavodoxin which can be used by an α-ketoglutaratesynthase and an electron donor regeneration system. For example, inEscherichia coli, the fdx gene exists as a gene encoding a 2Fe-2Scluster-containing ferredoxin (Ta, D. T. and Vickery, L. E. 1992. J.Biol. Chem. 267:11120-11125) and the yfhL gene is presumed to be a geneencoding a 4Fe-4S cluster-containing ferredoxin. Moreover, as flavodoxingenes, the fldA gene (Osborne, C. et al. 1991. J. Bacteriol. 173:1729-1737) and the fldB gene (Gaudu, P. and Weiss, B. 2000. J.Bacteriol. 182:1788-1793) are known to be present. In the genomicsequence of Corynebacterium glutamicum (GenBank Accession No. BA00036),the ferredoxin gene fdx (GenBank Accession No. BAB97942) is identifiedin the region from base position 562643 to 562963 and the fer gene(GenBank Accession No. BAB98495) is identified in the region from baseposition 1148953 to 1149270. Moreover, in Chlorobium tepidum, manyferredoxin genes exist and the genes for ferredoxin I and ferredoxin IIhave been identified as genes for 4Fe-4S-type ferredoxins which would beelectron acceptors for a pyruvate synthase (Yoon, K. S. et al. 2001. J.Biol. Chem. 276: 44027-44036). Ferredoxin genes or flavodoxin genesderived from bacteria possessing the reductive TCA cycle, such as theferredoxin gene in Hydrogenobacter thermophilus, may also be used.

Specific examples of a ferredoxin gene in Escherichia coli include thefdx gene shown in SEQ ID NO: 61 and located in the region from baseposition 2654770 to U.S. Pat. No. 2,655,105 (complementary strand) inthe genomic sequence of the Escherichia coli strain K-12 (GenBankAccession No. U00096), and the yfhL gene shown in SEQ ID NO: 63 andlocated in the region from base position 2697685 to 2697945 in the same.The amino acid sequences of the Fdx protein and the YfhL protein encodedby the same genes are shown in SEQ ID NO: 62 and SEQ ID NO: 64 (GenBankAccession Nos. AAC75578 and AAC75615, respectively). Specific examplesof a flavodoxin gene in Escherichia coli include the fldA gene shown inSEQ ID NO: 65 and located in the region from base position 710688 to710158 (complementary strand) in the genomic sequence of the Escherichiacoli strain K-12 (GenBank Accession No. U00096), and the fldB gene shownin SEQ ID NO: 67 and located in the region from base position 3037877 to3038398 in the same. The amino acid sequences of the fldA protein andthe fldB protein encoded by the same genes are shown in SEQ ID NO: 66and SEQ ID NO: 68 (GenBank Accession Nos. AAC73778 and AAC75933,respectively). Specific examples of a ferredoxin gene in Chlorobiumtepidum include the ferredoxin I gene shown in SEQ ID NO: 69 and locatedin the region from base position 1184078 to 1184266 in the genomicsequence of Chlorobium tepidum (GenBank Accession No. NC 002932) and theferredoxin II gene shown in SEQ ID NO: 71 and located in base position1184476 to 1184664 in the same. The amino acid sequences of ferredoxin Iand ferredoxin II encoded by the same genes are shown in SEQ ID NO: 70and SEQ ID NO: 72 (GenBank Accession Nos. AAM72491 and AAM72490,respectively). Moreover, examples of a ferredoxin gene include theferredoxin gene in Hydrogenobacter thermophilus (GenBank Accession No.BAE02673) and the ferredoxin gene represented by the region from baseposition 2345414 to 2345728 in the genomic sequence of Sulfolobussolfataricus.

Based on the homology with the above-described genes, as a ferredoxingene or flavodoxin gene, ferredoxin genes or flavodoxin genes clonedfrom bacteria in the genera Chlorobium, Desulfobacter, Aquifex,Hydrogenobacter, Thermoproteus, Pyrobaculum and the like may be used andferredoxin genes or flavodoxin genes cloned from bacteria inγ-Proteobacteria such as the genera Enterobacter, Klebsiella, Serratia,Erwinia, and Yersinia; coryneform bacteria such as Corynebacteriumglutamicum and Brevibacterium lactofermentum; Pseudomonas bacteria suchas Pseudomonas aeruginosa; Mycobacterium bacteria such as Mycobacteriumtuberculosis; and the like may be used.

The increase in the ability to produce ferredoxin and/or flavodoxin canbe confirmed, for example, by measuring the expression level (the amountof mRNA or protein) or the activity of ferredoxin and/or flavodoxin. Theactivity of ferredoxin and flavodoxin each can be measured using asuitable redox reaction system. For example, the activity of ferredoxincan be measured by reducing ferredoxin with a ferredoxin-NADP⁺ reductaseand determining the amount of cytochrome c reduced by the resultantreduced ferredoxin (Boyer, M. E. et al. 2006. Biotechnol. Bioeng. 94:128-138). Moreover, the activity of flavodoxin can be similarlymeasured.

Moreover, the microorganism of the present invention may have beenmodified to have a reduced α-ketoglutarate dehydrogenase (also referredto as “α-KGDH”) activity. The term “α-ketoglutarate dehydrogenase”refers to an enzyme which catalyzes a reaction for the production ofsuccinyl-CoA through the oxidative decarboxylation of α-ketoglutaricacid (2-oxoglutaric acid). Moreover, the activity to catalyze the samereaction is also referred to as “α-ketoglutarate dehydrogenaseactivity”. α-KGDH is also referred to as oxoglutarate dehydrogenase or2-oxoglutarate dehydrogenase.

The above-described reaction is catalyzed by three types of enzymes:α-KGDH (E1o; EC 1.2.4.2), dihydrolipoamide-S-succinyltransferase (E2o;EC 2.3.1.61), and dihydrolipoamide dehydrogenase (E3; EC 1.8.1.4). Thatis, these three types of enzymes catalyze the respective reactions belowand the α-KGDH activity specifically refers to an activity to catalyze areaction composed of these three reactions:

2-oxoglutarate+[dihydrolipoyllysine-residue succinyltransferase]lipoyllysine→[dihydrolipoyllysine-residue succinyltransferase]S-succinyldihydrolipoyllysine+CO₂,  E1o:

CoA+enzyme N6-(S-succinyldihydrolipoyl)lysine→succinyl-CoA+enzymeN6-(dihydrolipoyl)lysine,  E2o:

protein N6-(dihydrolipoyl)lysine+NAD⁺→proteinN6-(lipoyl)lysine+NADH+H⁺.  E3:

In Enterobacteriaceae, such as Pantoea ananatis, the subunit proteinsE1o, E2o, and E3 having these three respective enzymatic activities forma complex. These respective subunits are encoded by the sucA, sucB, andlpdA genes, and the sucA and sucB genes are located downstream of thesuccinate dehydrogenase iron-sulfur protein gene (sdhB) (U.S. Pat. No.6,331,419). Although, in the patent document, these genes are describedto be genes in Enterobacter agglomerans AJ13355, this bacterial strainwas reclassified later as Pantoea ananatis. As examples of genesencoding an α-KGDH of an enteric bacterium, the nucleotide sequences ofthe sucA, sucB, and lpdA genes in Pantoea ananatis AJ13355 are shown inSEQ ID NOs: 73, 75, and 77, respectively. Moreover, the amino acidsequences of the SucA, SucB, and LpdA proteins encoded by the same genesare shown in SEQ ID NOs: 74, 76, and 78, respectively. Moreover, theSucA, SucB, and LpdA proteins encoded by a-KGDH genes in Escherichiacoli, that is, the sucA, sucB, and lpdA genes, are disclosed as GenBankNP_415254, NP_415255, and NP_414658, respectively.

Moreover, in a coryneform bacterium, the E1o subunit is encoded by theodhA gene (also referred to as sucA gene; registered as NCgl1084 inGenBank Accession No. NC_003450) and the E3 subunit is encoded by thelpd gene (GenBank Accession No. Y16642). Meanwhile, the E2o subunit ispresumed to be encoded together with the E1o subunit by the odhA gene asa part of a bifunctional protein (see Usuda, Y. et al., Microbiology1996. 142: 3347-3354) or to be encoded by a gene different from the odhAgene and registered as NCgl2126 in GenBank Accession No. NC_003450.Accordingly, in the present invention, though the odhA gene is a geneencoding the E1o subunit, it may also encode the E1o and E2o subunitstogether. The nucleotide sequence of the odhA gene in Brevibacteriumlactofermentum ATCC 13032 and the amino acid sequence of the E1o subunitencoded by the same gene (WO2006/028298) are shown in SEQ ID NOs: 79 and80, respectively. Moreover, the nucleotide sequence of the lpd gene ofthe same and the amino acid sequence of the E3 subunit encoded by thesame gene (WO2006/028298) are shown in SEQ ID NOs: 81 and 82,respectively. Moreover, the nucleotide sequence of NCgl2126 with theabove-described GenBank Accession No. NC_003450 and the amino acidsequence of the protein encoded by the same sequence are shown in SEQ IDNOs: 83 and 84, respectively.

The reduction in the α-KGDH activity can be confirmed by measuring theα-KGDH activity with a known method (Shiio, I. and Ujigawa-Takeda, K.1980. Agric. Biol. Chem. 44: 1897-1904).

Moreover, the microorganism of the present invention may have beenmodified to have an increased ability to produce malyl-CoA. The term “anability to produce malyl-CoA” as used herein refers to an ability toperform the biosynthesis of malyl-CoA and does not require the generatedmalyl-CoA to be accumulated inside or outside microbial cells as aproduct. That is, for example, the generated malyl-CoA may be consumedimmediately.

The ability to produce malyl-CoA can be increased, for example, by (I)or (II) below:

(I) modifying a microorganism to have increased activities of enzyme(s)for synthesizing malyl-CoA from L-malic acid, malyl-CoA lyase, andisocitrate lyase; or

(II) modifying a microorganism to have increased activities of enzyme(s)for synthesizing malyl-CoA from L-malic acid, malyl-CoA lyase,glyoxylate carboligase, and 2-hydroxy-3-oxopropionate reductase and/orhydroxypyruvate reductase (WO2013/018734).

The term “enzyme for synthesizing malyl-CoA from L-malic acid” refers toa protein having an activity to catalyze a reaction in which L-malicacid is converted to malyl-CoA through the association with CoA.Examples of an enzyme for synthesizing malyl-CoA from L-malic acidinclude malate thiokinase, succinyl-CoA synthase, andsuccinyl-CoA:malate-CoA-transferase. In the present invention, theactivity of one or more types of enzymes selected from enzymes forsynthesizing malyl-CoA from L-malic acid may be increased. That is, forexample, the activity of any of malate thiokinase, succinyl-CoAsynthase, and succinyl-CoA:malate-CoA-transferase may be increased, orthe activity of all of them may be increased. The activity of a proteincan be increased, for example, by increasing the expression of a geneencoding the same protein. Detailed procedures to increase the activityof a protein will be described below.

The term “malate thiokinase” refers to an enzyme which reversiblycatalyzes a reaction for the production of malyl-CoA from L-malic acidand CoA (EC 6.2.1.9). Moreover, the activity to catalyze the samereaction is also referred to as “malate thiokinase activity”.Additionally, the above-described reaction is known to be reversible invivo and ex vivo, and that is, malate thiokinase is known to be able tocatalyze the reverse reaction of the above-described reaction as well.Malate thiokinase is also referred to as malyl-CoA synthase, malate-CoAligase, or malyl-coenzyme A synthase.

The malate thiokinase is known to function in a multisubunit complex,usually a complex composed of an α-subunit and a β-subunit. Theα-subunit is encoded by the mtkB gene and the β-subunit is encoded bythe mtkA gene. The mtkA and mtkB genes are usually located sequentiallyon the genome.

Genes encoding malate thiokinases have been identified in organismspossessing an assimilation pathway for C1-carbon sources such as methane(J. Bacteriol., 176(23), 7398-7404 (1994)) and organisms possessing the3-hydroxypropionate pathway (Arch. Microbiol., 151, 252-256 (1989)).Additionally, in general, there is the mclA gene encoding the malyl-CoAlyase described below in close proximity of the mtkAB genes on thegenome encoding a malate thiokinase. Examples of a species in which themkAB genes and the mclA gene are located in close proximity on thegenome can be identified by, for example, NCBI BLAST(http://www.ncbi.nlm.nih.gov/BLAST/).

Specific examples of genes encoding a malate thiokinase include, forexample, the mtkAB genes in Methylobacterium bacteria such asMethylobacterium extorquens; Mesorhizobium bacteria such asMesorhizobium loti; Granulibacter bacteria such as Granulibacterbethesdensis; Roseobacter bacteria such as Roseobacter denitrificans;Moorella bacteria such as Moorella thermoacetica; Hyphomicrobiumbacteria such as Hyphomicrobium methylovorum; Chloroflexus bacteria suchas Chloroflexus aurantiacus; Nitrosomonas bacteria such as Nitrosomonaseuropaea; and Methylococcus bacteria such as Methylococcus capsulatus.

The entire nucleotide sequence of the genomic DNA of theMethylobacterium extorquens strain AM1 has been known (GenBank accessionnumber NC 012808.1) and, furthermore, the nucleotide sequences of themtkAB genes encoding the malate thiokinase in the Methylobacteriumextorquens strain AM1 have been reported. That is, the mtkA gene in theMethylobacterium extorquens strain AM1 corresponds to a sequence frombase position 1803549 to 1804721 in the genomic sequence of theMethylobacterium extorquens strain AM1 described in GenBank accessionnumber NC_012808.1. Moreover, the mtkB gene in the Methylobacteriumextorquens strain AM1 corresponds to a sequence from base position1804744 to 1805634 in the genomic sequence of the Methylobacteriumextorquens strain AM1 described in GenBank accession number NC_012808.1.The nucleotide sequence of the mtkA gene in the Methylobacteriumextorquens strain AM1 and the amino acid sequence of the protein encodedby the same gene are shown in SEQ ID NOs: 99 and 100, respectively. Thenucleotide sequence of the mtkB gene in the Methylobacterium extorquensstrain AM1 and the amino acid sequence of the protein encoded by thesame gene are shown in SEQ ID NOs: 101 and 102, respectively.

The entire nucleotide sequence of the genomic DNA of the Mesorhizobiumloti strain MAFF303099 has been known (GenBank accession numberNC_002678.2) and, furthermore, the nucleotide sequences of the mtkABgenes encoding the malate thiokinase in the Mesorhizobium loti strainMAFF303099 have been reported. That is, the mtkA gene and the mtkB genein the Mesorhizobium loti strain MAFF303099 correspond to a sequencefrom base position 1110720 to 1111904 and a sequence from base position1111919 to 1112818 in the genomic sequence of the Mesorhizobium lotistrain MAFF303099 (GenBank accession number NC_002678.2), respectively.The nucleotide sequence of the mtkA gene in the Mesorhizobium lotistrain MAFF303099 and the amino acid sequence of the protein encoded bythe same gene are shown in SEQ ID NOs: 103 and 104, respectively. Thenucleotide sequence of the mtkB gene in the Mesorhizobium loti strainMAFF303099 and the amino acid sequence of the protein encoded by thesame gene are shown in SEQ ID NOs: 105 and 106, respectively.

The entire nucleotide sequence of the genomic DNA of the Granulibacterbethesdensis strain CGDNIH1 has been known (GenBank accession numberNC_008343.1) and, furthermore, the nucleotide sequences of the mtkABgenes encoding the malate thiokinase in the Granulibacter bethesdensisstrain CGDNIH1 have been reported. That is, the mtkA gene and the mtkBgene in the Granulibacter bethesdensis strain CGDNIH1 correspond to asequence from base position 55236 to 56405 and a sequence from baseposition 56421 to 57317 in the genomic sequence of the Granulibacterbethesdensis strain CGDNIH1 (GenBank accession number NC_008343.1),respectively. The nucleotide sequence of the mtkA gene in theGranulibacter bethesdensis strain CGDNIH1 and the amino acid sequence ofthe protein encoded by the same gene are shown in SEQ ID NOs: 107 and108, respectively. The nucleotide sequence of the mtkB gene in theGranulibacter bethesdensis strain CGDNIH1 and the amino acid sequence ofthe protein encoded by the same gene are shown in SEQ ID NOs: 109 and110, respectively.

Any malate thiokinase genes can be used without any particularlimitation as long as they encode proteins which function in a host. Forexample, genes encoding malate thiokinases of Hyphomicrobiummethylovolum, Hyphomicrobium denitrificans, the Rhizobium sp. strainNGR234, Granulibacter bethesdensis, Nitrosomonas europaea, andMethylococcus capsulatus have been reported to be expressed and functionin E. coli, Pantoea ananatis, and Corynebacterium glutamicum(WO2013/018734).

The α-subunit and the β-subunit of the malate thiokinase are highlyhomologous to the α-subunit and the β-subunit of a succinyl-CoA synthaseas described below, respectively. As indicated in Examples sectionbelow, the inventors found that a succinyl-CoA synthase shows the malatethiokinase activity. It means that the malate thiokinase activity canalso be increased by increasing the succinyl-CoA synthase activity.

The increase in the malate thiokinase activity can be confirmed, forexample, by preparing crude enzyme solutions from microorganisms beforeand after the modification and comparing the malate thiokinase activityin the crude enzyme solutions. The malate thiokinase activity can bemeasured according to, for example, a method of Louis (Louis B. Hersh JBiol Chem. 1973 Nov. 10; 248(21):7295-303). Specifically, the malatethiokinase activity can be determined by adding L-malic acid to areaction liquid containing phenylhydrazine, CoA, ATP, malyl-CoA lyase,and a crude enzyme solution, wherein phenylhydrazine quickly reacts withglyoxylic acid and gives a color, and measuring spectroscopically theamount of generated glyoxylate phenylhydrazine. This method utilizes aphenomenon that malyl-CoA generated by malate thiokinase is cleaved bymalyl-CoA lyase into acetyl-CoA and glyoxylic acid.

The term “succinyl-CoA synthase” refers to an enzyme which catalyzes areaction for the production of succinyl-CoA from succinic acid andcoenzyme A (hereinafter referred to as CoA) accompanied by a hydrolysisreaction of nucleotide triphosphate, such as ATP or GTP, to nucleotidediphosphate and inorganic phosphate (EC 6.2.1.5 or EC 6.2.1.4).Moreover, the activity to catalyze the same reaction is also referred toas “succinyl-CoA synthase activity”. Additionally, the above-describedreaction is known to be reversible in vivo and ex vivo, and that is, thesuccinyl-CoA synthase is known to be able to catalyze the reversereaction of the above-described reaction as well. Succinyl-CoA synthaseis also referred to as succinyl-CoA ligase, succinyl-coenzyme Asynthase, succinate thiokinase, succinic thiokinase, succinatephosphorylating enzyme, or P-enzyme.

The succinyl-CoA synthase is known to function in a multisubunitcomplex, usually a complex composed of an α-subunit and a β-subunit. Thea-subunit is encoded by the sucD gene and the β-subunit is encoded bythe sucC gene. The sucC and sucD genes are usually located sequentiallyon the genome.

The presence of genes encoding a succinyl-CoA synthase has beenidentified in various organisms. Genes encoding succinyl-CoA synthaseshave been registered in, for example, various databases such as KEGG(Kyoto Encyclopedia of Genes and Genomes; http://www.genome.jp/kegg/),NCBI (National Center for Biotechnology Information;http://www.ncbi.nlm.nih.gov/gene/), and BRENDA (BRaunschweig ENzymeDAtabase; http://www.brenda-enzymes.info/). Any succinyl-CoA synthasegenes can be used without any particular limitation as long as theyencode proteins which function in a host. Endogenous succinyl-CoAsynthase genes in a host microorganism may also be used in terms of, forexample, the efficiency of the succinyl-CoA production.

Specific examples of genes encoding a succinyl-CoA synthase include, forexample, the sucCD genes of Escherichia bacteria such as Escherichiacoli; Pantoea bacteria such as Pantoea ananatis; and Corynebacteriumbacteria such as Corynebacterium glutamicum, Corynebacterium efficiens,Corynebacterium ammoniagenes.

The entire nucleotide sequence of the genomic DNA of the Escherichiacoli strain MG1655 has been known (GenBank accession number NC_000913.3)and, furthermore, the nucleotide sequences of the sucCD genes encodingthe succinyl-CoA synthase in the Escherichia coli strain MG1655 havebeen reported. That is, the sucC gene corresponds to a sequence frombase position 762237 to 763403 in the genomic sequence of theEscherichia coli strain MG1655 described in GenBank accession numberNC_000913.3. Moreover, the sucD gene corresponds to a sequence from baseposition 763403 to 764272 in the genomic sequence of the Escherichiacoli strain MG1655 described in GenBank accession number NC_000913.3.The nucleotide sequence of the sucC gene in the Escherichia coli strainMG1655 and the amino acid sequence of the protein encoded by the samegene are shown in SEQ ID NOs: 111 and 112, respectively. The nucleotidesequence of the sucD gene in the Escherichia coli strain MG1655 and theamino acid sequence of the protein encoded by the same gene are shown inSEQ ID NOs: 113 and 114, respectively.

The entire nucleotide sequence of the genomic DNA of the Pantoeaananatis strain AJ13355 has been known (GenBank accession numberNC_017531.1) and, furthermore, the nucleotide sequences of the sucCDgenes encoding the succinyl-CoA synthase in the Pantoea ananatis strainAJ13355 have been reported. That is, the sucC gene corresponds to asequence from base position 610188 to 611354 in the genomic sequence ofthe Pantoea ananatis strain AJ13355 described in GenBank accessionnumber NC_017531.1. Moreover, the sucD gene corresponds to a sequencefrom base position 611354 to 612229 in the genomic sequence of thePantoea ananatis strain AJ13355 described in GenBank accession numberNC_017531.1. The nucleotide sequence of the sucC gene in the Pantoeaananatis strain AJ13355 and the amino acid sequence of the proteinencoded by the same gene are shown in SEQ ID NOs: 115 and 116,respectively. The nucleotide sequence of the sucD gene in the Pantoeaananatis strain AJ13355 and the amino acid sequence of the proteinencoded by the same gene are shown in SEQ ID NOs: 117 and 118,respectively.

The entire nucleotide sequence of the genomic DNA of the Corynebacteriumglutamicum strain ATCC13032 has been known (GenBank accession numberNC_003450.3) and, furthermore, the nucleotide sequences of the sucCDgenes encoding the succinyl-CoA synthase in the Corynebacteriumglutamicum strain ATCC13032 have been reported. That is, the sucC genecorresponds to a sequence complementary to a sequence from base position2725382 to 2726578 in the genomic sequence of the Corynebacteriumglutamicum strain ATCC13032 described in GenBank accession numberNC_003450.3. Moreover, the sucD gene corresponds to a sequencecomplementary to a sequence from base position 2724476 to 2725360 in thegenomic sequence of the Corynebacterium glutamicum strain ATCC13032described in GenBank accession number NC_003450.3. The nucleotidesequence of the sucC gene in the Corynebacterium glutamicum strainATCC13032 and the amino acid sequence of the protein encoded by the samegene are shown in SEQ ID NOs: 119 and 120, respectively. The nucleotidesequence of the sucD gene in the Corynebacterium glutamicum strainATCC13032 and the amino acid sequence of the protein encoded by the samegene are shown in SEQ ID NOs: 121 and 122, respectively. The nucleotidesequence of the sucC gene in the Corynebacterium glutamicum strain 2256(ATCC 13869) and the amino acid sequence of the protein encoded by thesame gene are shown in SEQ ID NOs: 123 and 124, respectively. Thenucleotide sequence of the sucD gene in the Corynebacterium glutamicumstrain 2256 (ATCC 13869) and the amino acid sequence of the proteinencoded by the same gene are shown in SEQ ID NOs: 125 and 126,respectively.

Additionally, the succinyl-CoA synthase activity and/or malatethiokinase activity may be increased by introducing a mutation into asuccinyl-CoA synthase. Examples of a mutation which increases at leastthe malate thiokinase activity include, for example, the mutationsbelow:

a mutation substituting proline at position 124 with alanine in theα-subunit encoded by the sucD gene of Escherichia coli;

a mutation substituting tyrosine at position 157 with glycine in theα-subunit encoded by the sucD gene of Escherichia coli;

a mutation substituting valine at position 161 with alanine in theα-subunit encoded by the sucD gene of Escherichia coli;

a mutation substituting glutamic acid at position 97 with aspartic acidin the α-subunit encoded by the sucD gene of Escherichia coli; and

a mutation substituting glycine at position 271 with alanine in theβ-subunit encoded by the sucC gene of Escherichia coli.

Only one of these mutations may be introduced, or two or more of themutations may be introduced. For example, mutant succinyl-CoA synthasegenes may be constructed, which has alanine residues substituted forvaline at position 161 in the α-subunit encoded by the sucD gene ofEscherichia coli and for glycine at position 271 in the β-subunitencoded by the sucC gene.

A succinyl-CoA synthase having none of the above-described mutations isreferred to as “wild-type succinyl-CoA synthase” and a gene encoding awild-type succinyl-CoA synthase is likewise referred to as “wild-typesuccinyl-CoA synthase gene”. Moreover, a succinyl-CoA synthase havingany of the above-described mutations is referred to as “mutantsuccinyl-CoA synthase” and a gene encoding a mutant succinyl-CoAsynthase is likewise referred to as “mutant succinyl-CoA synthase gene”.

The wild-type succinyl-CoA synthase is not limited to the wild-typesuccinyl-CoA synthase of Escherichia coli as indicated above and mayalso be a conservative variant thereof. Additionally, the position ofmutation in the notation for each of the above-described mutations is arelative value and is variable depending on amino acid deletion,insertion, addition, or the like. For example, “valine at position 161in the α-subunit” means an amino acid residue corresponding to thevaline residue at position 161 in SEQ ID NO: 114. That is, “valine atposition 161 in the α-subunit” is intended to be the 160th amino acidresidue from the N-terminus in cases where one amino acid residue isdeleted in a region on the N-terminal side to the position 161.Moreover, “valine at position 161 in the α-subunit” is intended to bethe 162th amino acid residue from the N-terminus in cases where oneamino acid residue is inserted to a region on the N-terminal side to theposition 161.

The amino acid residues targeted to the above-described mutations in anarbitrary amino acid sequence can be determined by aligning thearbitrary amino acid sequence to the amino acid sequence of SEQ ID NO:114. The alignment can be performed, for example, by using a known geneanalysis software program. Specific examples of the software programinclude DNASIS produced by Hitachi Solutions, Ltd., GENETYX produced byGENETYX Co., and the like (Elizabeth C. Tyler et al., Computers andBiomedical Research, 24(1), 72-96, 1991; Barton G J et al., Journal ofmolecular biology, 198(2), 327-37. 1987).

The increase in the succinyl-CoA synthase activity can be confirmed, forexample, by preparing crude enzyme solutions from microorganisms beforeand after the modification and comparing the succinyl-CoA synthaseactivity in the crude enzyme solutions. The succinyl-CoA synthaseactivity can be measured according to, for example, a method ofWilliamson (John R. Williamson, Barbara E. Corkey Methods in Enzymology,edited by Colowich J M. New York: Academic, 1969, p. 434-514).Specifically, the succinyl-CoA synthase activity can be determined byadding succinic acid to a reaction liquid containing CoA, ATP,phosphoenolpyruvic acid, pyruvate kinase, lactate dehydrogenase, NADH,and a crude enzyme solution and measuring spectroscopically the amountof consumed NADH.

The term “succinyl-CoA:malate-CoA-transferase” refers to an enzyme whichcatalyzes a reaction for the production of succinic acid and malyl-CoAfrom succinyl-CoA and L-malic acid (EC 2.8.3.-). Moreover, the activityto catalyze the same reaction is also referred to as“succinyl-CoA:malate-CoA-transferase activity”.Succinyl-CoA:malate-CoA-transferase is also referred to assuccinyl-CoA(S)-malate-CoA-transferase, or L-carnitine dehydratase/bileacid-inducible protein family.

Known examples of a succinyl-CoA:malate-CoA-transferase include asuccinyl-CoA:malate-CoA-transferase which functions in a multisubunitcomplex. Such a succinyl-CoA:malate-CoA-transferase is usually composedof a subunit encoded by the smtA gene and a subunit encoded by the smtBgene. The smtA and smtB genes are usually located sequentially on thegenome.

Specific examples of genes encoding such asuccinyl-CoA:malate-CoA-transferase include, for example, the smtABgenes in Chloroflexus bacteria such as Chloroflexus aurantiacus andAccumulibacter bacteria such as Accumulibacter phosphatis, and homologsthereof. The proteins encoded by the smtA gene and the smtB gene arehighly homologous to each other and, for example, proteins encoded bythe smtA gene and the smtB gene of Chloroflexus aurantiacus have anamino acid homology of 59%. Additionally, the smtAB genes ofChloroflexus aurantiacus have been reported to be expressed and functionin E. coli (Friedmann S et al. (2006) J Bacteriol. 188(7):2646-55).

Moreover, examples of a succinyl-CoA:malate-CoA-transferase also includea succinyl-CoA:malate-CoA-transferase encoded by a single gene. Such asuccinyl-CoA:malate-CoA-transferase is not particularly limited as longas it is classified into CoA-transferase family III (CaiB/BaiF) and hasthe succinyl-CoA:malate-CoA-transferase activity.

Specific examples of a gene encoding such asuccinyl-CoA:malate-CoA-transferase include, for example, the smtB genehomologs in Magnetospirillum bacteria such as Magnetospirillummagneticum and Rhodospirillum bacteria such as Rhodospirillum rubrum.Such a gene encoding a succinyl-CoA:malate-CoA-transferase is alsoreferred to as “smt gene”.

The entire nucleotide sequence of the genomic DNA of the Chloroflexusaurantiacus strain J-10-fl has been known (GenBank accession numberNC_010175.1) and, furthermore, the nucleotide sequences of the smtABgenes encoding the succinyl-CoA:malate-CoA-transferase of theChloroflexus aurantiacus strain J-10-fl (hereinafter also referred to as“Ca_smtAB genes”) have been reported. That is, the Ca_smtA gene and theCa_smtB gene correspond to a sequence complementary to a sequence frombase position 224515 to 225882 and a sequence complementary to asequence from base position 223035 to 224252 in the genomic sequence ofthe Chloroflexus aurantiacus strain J-10-fl (GenBank accession numberNC_010175.1), respectively. The nucleotide sequence of the Ca_smtA geneand the amino acid sequence of the protein encoded by the same gene(YP_001633822) are shown in SEQ ID NOs: 127 and 128, respectively. Thenucleotide sequence of the Ca_smtB gene and the amino acid sequence ofthe protein encoded by the same gene (YP_001633821) are shown in SEQ IDNOs: 129 and 130, respectively.

The entire nucleotide sequence of the genomic DNA of the CandidatusAccumulibacter phosphatis clade IIA strain UW-1 has been known (GenBankaccession number NC_013194.1). Examples of thesuccinyl-CoA:malate-CoA-transferase genes in the CandidatusAccumulibacter phosphatis clade IIA strain UW-1 include the homologs ofthe Ca_smtA gene and the Ca_smtB gene (hereinafter also referred to as“Ap_smtA gene” and “Ap_smtB gene”, respectively; as well as collectivelyreferred to as “Ap_smtAB genes”). The Ap_smtA gene and the Ap_smtB genecorrespond to a sequence from base position 2888316 to 2889563 and asequence from base position 2889587 to 2890813 in the genomic sequenceof the Candidatus Accumulibacter phosphatis clade IIA strain UW-1(GenBank accession number NC_013194.1), respectively. The nucleotidesequence of the Ap_smtA gene and the amino acid sequence of the proteinencoded by the same gene are shown in SEQ ID NOs: 131 and 132,respectively. The nucleotide sequence of the Ap_smtB gene and the aminoacid sequence of the protein encoded by the same gene are shown in SEQID NOs: 133 and 134, respectively.

The entire nucleotide sequence of the genomic DNA of the Rhodospirillumrubrum strain ATCC 11170 has been known (GenBank accession numberNC_007643.1). Examples of the succinyl-CoA:malate-CoA-transferase genesin the Rhodospirillum rubrum strain ATCC 11170 include a gene homologousto the Ca_smtB gene (hereinafter also referred to as “Rr_smt gene”). TheRr_smt gene corresponds to a sequence complementary to a sequence frombase position 2965790 to 2967016 in the genomic sequence of theRhodospirillum rubrum strain ATCC 11170 (GenBank accession numberNC_007643.1). The nucleotide sequence of the Rr_smt gene and the aminoacid sequence of the protein encoded by the same gene (YP_427637) areshown in SEQ ID NOs: 135 and 136, respectively.

The entire nucleotide sequence of the genomic DNA of theMagnetospirillum magneticum strain AMB-1 has been known (GenBankaccession number NC_007626.1). Examples of thesuccinyl-CoA:malate-CoA-transferase gene in the Magnetospirillummagneticum strain AMB-1 include a gene homologous to the Ca_smtB gene(hereinafter also referred to as “Mm_smt gene”). The Mm_smt genecorresponds to a sequence complementary to a sequence from base position2307230 to 2308438 in the genomic sequence of the Magnetospirillummagneticum strain AMB-1 (GenBank accession number NC_007626.1). Thenucleotide sequence of the Mm_smt gene and the amino acid sequence ofthe protein encoded by the same gene (YP_421496) are shown in SEQ IDNOs: 137 and 138, respectively.

The increase in the succinyl-CoA:malate-CoA-transferase activity can beconfirmed, for example, by preparing crude enzyme solutions frommicroorganisms before and after the modification and comparing thesuccinyl-CoA:malate-CoA-transferase activity in the crude enzymesolutions. The succinyl-CoA:malate-CoA-transferase activity can bemeasured according to, for example, a method of Friedmann (Friedmann Set al. (2006) J Bacteriol. 188(7):2646-55). Specifically, thesuccinyl-CoA:malate-CoA-transferase activity can be determined by addingL-malic acid to a reaction liquid containing phenylhydrazine,succinyl-CoA, malyl-CoA lyase, and a crude enzyme solution, whereinphenylhydrazine quickly reacts with glyoxylic acid and gives a color,and measuring spectroscopically the amount of generated glyoxylatephenylhydrazine.

The term “malyl-CoA lyase” refers to an enzyme which reversiblycatalyzes a reaction for the production of acetyl-CoA and glyoxylic acidfrom malyl-CoA (EC 4.1.3.24). Moreover, the activity to catalyze thesame reaction is also referred to as “malyl-CoA lyase activity”.Malyl-CoA lyase is also referred to as malyl-coenzyme A lyase, or (3S)-3-carboxy-3-hydroxypropanoyl-CoA glyoxylate-lyase.

Specific examples of a gene encoding a malyl-CoA lyase include, forexample, the mclA genes in Methylobacterium bacteria such asMethylobacterium extorquens; Mesorhizobium bacteria such asMesorhizobium loti; Granulibacter bacteria such as Granulibacterbethesdensis; Roseobacter bacteria such as Roseobacter denitrificans;Moorella bacteria such as Moorella thermoacetica; Hyphomicrobiumbacteria such as Hyphomicrobium methylovorum; Chloroflexus bacteria suchas Chloroflexus aurantiacus; Nitrosomonas bacteria such as Nitrosomonaseuropaea; and Methylococcus bacteria such as Methylococcus capsulatus.

The mclA gene encoding the malyl-CoA lyase in the Methylobacteriumextorquens strain AM1 corresponds to a sequence from base position1808790 to 1809764 in the genomic sequence of the Methylobacteriumextorquens strain AM1 described in GenBank accession number NC_012808.1.The nucleotide sequence of the mclA gene in the Methylobacteriumextorquens strain AM1 and the amino acid sequence of the protein encodedby the same gene are shown in SEQ ID NOs: 139 and 140, respectively.

The mclA gene encoding the malyl-CoA lyase in the Mesorhizobium lotistrain MAFF303099 corresponds to a sequence from base position 1109744to 1110700 in the genomic sequence of the Mesorhizobium loti strainMAFF303099 (GenBank accession number NC_002678.2). The nucleotidesequence of the mclA gene in the Mesorhizobium loti strain MAFF303099and the amino acid sequence of the protein encoded by the same gene areshown in SEQ ID NOs: 141 and 142, respectively.

The DNA sequence of the mclA gene encoding the malyl-CoA lyase in theGranulibacter bethesdensis strain CGDNIH1 corresponds to a sequence frombase position 60117 to 61112 in the genomic sequence of theGranulibacter bethesdensis strain CGDNIH1 (GenBank accession numberNC_008343.1). The nucleotide sequence of the mclA gene in theGranulibacter bethesdensis strain CGDNIH1 and the amino acid sequence ofthe protein encoded by the same gene are shown in SEQ ID NOs: 143 and144, respectively.

The increase in the malyl-CoA lyase activity can be confirmed, forexample, by preparing crude enzyme solutions from microorganisms beforeand after the modification and comparing the malyl-CoA lyase activity inthe crude enzyme solutions. The malyl-CoA lyase activity can be measuredaccording to, for example, a method of Louis (Louis B. Hersh J BiolChem. 1973 Nov. 10; 248(21):7295-303). Specifically, the malyl-CoA lyaseactivity can be determined by adding L-malic acid to a reaction liquidcontaining phenylhydrazine, CoA, ATP, malate thiokinase, and a crudeenzyme solution, wherein phenylhydrazine quickly reacts with glyoxylicacid and gives a color, and measuring spectroscopically the amount ofgenerated glyoxylate phenylhydrazine. This method utilizes a phenomenonthat malyl-CoA generated by malate thiokinase is cleaved by malyl-CoAlyase into acetyl-CoA and glyoxylic acid. Alternatively, the malyl-CoAlyase activity can be determined similarly using malyl-CoA instead ofthe combination of CoA, ATP, malate thiokinase, and L-malic acid.

The term “isocitrate lyase” refers to an enzyme which reversiblycatalyzes a reaction for the production of glyoxylic acid and succinicacid from isocitric acid (EC 4.1.3.1). Moreover, the activity tocatalyze the same reaction is also referred to as “isocitrate lyaseactivity”. Isocitrate lyase is also referred to as isocitrase,isocitritase, isocitratase, threo-Ds-isocitrate glyoxylate-lyase, orisocitrate glyoxylate-lyase.

The presence of a gene encoding an isocitrate lyase has been identifiedin various organisms. Genes encoding isocitrate lyases have beenregistered in, for example, various databases such as KEGG (KyotoEncyclopedia of Genes and Genomes; http://www.genome.jp/kegg/), NCBI(National Center for Biotechnology Information;http://www.ncbi.nlm.nih.gov/gene/), and BRENDA (BRaunschweig ENzymeDAtabase; http://www.brenda-enzymes.info/). Any isocitrate lyase genescan be used without any particular limitation as long as they encodeproteins which function in a host. Endogenous isocitrate lyase gene in ahost microorganism may also be used in terms of, for example, theefficiency of the isocitrate lyase production.

Specific examples of a gene encoding an isocitrate lyase include, forexample, the aceA genes in Escherichia bacteria such as Escherichiacoli; Pantoea bacteria such as Pantoea ananatis; and Corynebacteriumbacteria such as Corynebacterium glutamicum.

The aceA gene encoding the isocitrate lyase in the Escherichia colistrain MG1655 corresponds to a sequence from base position 4215132 to4216436 in the genomic sequence of the Escherichia coli strain MG1655described in GenBank accession number NC_000913.3. The nucleotidesequence of the aceA gene in the Escherichia coli strain MG1655 and theamino acid sequence of the protein encoded by the same gene are shown inSEQ ID NOs: 145 and 146, respectively.

The aceA gene encoding the isocitrate lyase in the Pantoea ananatisstrain AJ13355 corresponds to a sequence from base position 4068278 to4069579 in the genomic sequence of the Pantoea ananatis strain AJ13355described in GenBank accession number NC_017531.1. The nucleotidesequence of the aceA gene in the Pantoea ananatis strain AJ13355 and theamino acid sequence of the protein encoded by the same gene are shown inSEQ ID NOs: 147 and 148, respectively.

Moreover, for example, some Corynebacterium bacteria have two copies ofisocitrate lyase genes (hereinafter also referred to as “ICL1 gene” and“ICL2 gene”). The ICL1 gene in the Corynebacterium glutamicum strainATCC13032 (Cgl2331) corresponds to a sequence from base position 2470741to 2472039 in the genomic sequence of the Corynebacterium glutamicumstrain ATCC13032 described in GenBank accession number NC_003450.3. TheICL2 gene in the Corynebacterium glutamicum strain ATCC13032 (Cgl0097)corresponds to a sequence complementary to a sequence from base position106392 to 105838 in the genomic sequence of the Corynebacteriumglutamicum strain ATCC13032 described in GenBank accession numberNC_003450.3. The nucleotide sequence of the ICL1 gene in theCorynebacterium glutamicum strain ATCC13032 and the amino acid sequenceof the protein encoded by the same gene are shown in SEQ ID NOs: 149 and150, respectively. The nucleotide sequence of the ICL2 gene in theCorynebacterium glutamicum strain ATCC13032 and the amino acid sequenceof the protein encoded by the same gene are shown in SEQ ID NOs: 151 and152, respectively. Moreover, the nucleotide sequence of the ICL1 gene inthe Corynebacterium glutamicum strain 2256 (ATCC 13869) and the aminoacid sequence of the protein encoded by the same gene are shown in SEQID NOs: 153 and 154, respectively. The nucleotide sequence of the ICL2gene in the Corynebacterium glutamicum strain 2256 (ATCC 13869) and theamino acid sequence of the protein encoded by the same gene are shown inSEQ ID NOs: 155 and 156, respectively.

The aceA gene is typically a component of an operon consisting of theaceBAK genes. As described below, the activity of a malate synthaseencoded by the aceB can be attenuated. Therefore, when the isocitratelyase activity is increased, the expression of the aceA gene may beincreased, for example, by deleting the aceB gene in the aceBAK operonand introducing a strong promoter at the same time, as described inExamples section.

The increase in the isocitrate lyase activity can be confirmed, forexample, by preparing crude enzyme solutions from microorganisms beforeand after the modification and comparing the isocitrate lyase activityin the crude enzyme solutions. The isocitrate lyase activity can bemeasured according to, for example, a method of Hoyt et al., (Hoyt J Cet al. (1988) Biochim Biophys Acta. 14; 966(1):30-5). Specifically, theisocitrate lyase activity can be determined by adding isocitric acid toa reaction liquid containing phenylhydrazine and a crude enzymesolution, wherein phenylhydrazine quickly reacts with glyoxylic acid andgives a color, and measuring spectroscopically the amount of generatedglyoxylate phenylhydrazine. Moreover, the isocitrate lyase activity canbe measured according to, for example, a method of Mackintosh et al.,(Mackintosh, C et al. (1988) Biochem. J. 250, 25-31). Specifically, theisocitrate lyase activity can be determined by adding glyoxylic acid andsuccinic acid to a reaction liquid containing NADP, isocitratedehydrogenase, and a crude enzyme solution and measuringspectroscopically the amount of generated NADPH.

The term “glyoxylate carboligase” refers to an enzyme which catalyzes areaction for converting two glyoxylic acid molecules to one2-hydroxy-3-oxopropionic acid molecule (EC 4.1.1.47). The reaction isaccompanied by decarboxylation of one carbon dioxide molecule. Examplesof a gene encoding a glyoxylate carboligase include, for example, thegcl genes in Corynebacterium bacteria such as Corynebacteriumglutamicum; Escherichia bacteria such as Escherichia coli; andRhodococcus bacteria such as Rhodococcus jostii.

The term “2-hydroxy-3-oxopropionate reductase” refers to an enzyme whichconverts 2-hydroxy-3-oxopropionic acid to glyceric acid by using NADH asan electron donor (EC 1.1.1.60). Examples of a gene encoding a2-hydroxy-3-oxopropionate reductase include, for example, the glxR genesin Corynebacterium bacteria such as Corynebacterium glutamicum andEscherichia bacteria such as Escherichia coli.

The term “hydroxypyruvate reductase” refers to an enzyme which convertshydroxypyruvic acid to glyceric acid by using NADH or NADPH as anelectron donor (EC 1.1.1.81). Examples of a gene encoding ahydroxypyruvate reductase include, for example, the ycdW genes inEscherichia bacteria such as Escherichia coli and Pantoea bacteria suchas Pantoea ananatis.

The genes used for these other modifications are not limited to thegenes exemplified above and genes having a known nucleotide sequence,and may be variants thereof, so long as they encode proteins of whichthe original functions are maintained. For the variants of genes andproteins, the descriptions for conservative variants of theabove-described dicarboxylic acid exporter proteins and the genesencoding them can be applied, mutatis mutandis.

The phrase “maintaining the original function” can mean that a variantof the protein has an activity which corresponds to the activity of theoriginal protein. That is, for example, “retaining the originalfunction” in terms of NADH dehydrogenase can mean that a protein has theNADH dehydrogenase activity and “retaining the original function” interms of malate: quinone oxidoreductase can mean that a protein has themalate: quinone oxidoreductase activity. Additionally, in cases where aprotein functions in a multisubunit complex, “retaining the originalfunction” in terms of each subunit may mean that each subunit forms acomplex with the remaining subunit(s) and the resultant complex has thecorresponding activity. That is, for example, “retaining the originalfunction” in terms of each subunit of NDH-1 may mean that each subunitforms a complex with the remaining subunits and the resultant complexhas the NDH-1 activity.

<1-4> Methods for Increasing Activity of Protein

Hereafter, methods for increasing the activity of a protein will beexplained.

The expression “the activity of a protein is increased” can mean thatthe activity of the protein per cell is increased as compared with thatof a non-modified strain such as a wild-type strain and parent strain.The state that “the activity of a protein is increased” can also beexpressed as “the activity of a protein is enhanced”. Specifically, theexpression “the activity of a protein is increased” can mean that thenumber of molecules of the protein per cell is increased, and/or thefunction of each molecule of the protein is increased as compared withthose of a non-modified strain. That is, the term “activity” in theexpression “the activity of a protein is increased” is not limited tothe catalytic activity of the protein, but may mean the transcriptionamount of a gene (the amount of mRNA) coding for the protein, or thetranslation amount of the gene (the amount of the protein). Furthermore,the state that “the activity of a protein is increased” includes notonly a state that the activity of an objective protein is increased in astrain inherently having the activity of the objective protein, but alsoa state that the activity of an objective protein is imparted to astrain not inherently having the activity of the objective protein.Furthermore, so long as the activity of the protein is eventuallyincreased, the activity of the objective protein inherently contained ina microorganism may be reduced or eliminated, and then an appropriatetype of the protein may be imparted thereto.

Although the degree of the increase in the activity of a protein is notparticularly limited so long as the activity of the protein is increasedas compared with a non-modified strain, the activity of the protein maybe increased, for example, 1.5 times or more, 2 times or more, or 3times or more, as compared with that of a non-modified strain.Furthermore, when the non-modified strain does not have the activity ofthe objective protein, it is sufficient that the protein is produced byintroducing the gene coding for the protein, and for example, theprotein may be produced to such an extent that the enzyme activity canbe measured.

The modification that increases the activity of a protein can beattained by, for example, increasing the expression of a gene coding forthe protein. The phrase that “the expression of a gene is increased” canalso be referred to as “the expression of a gene is enhanced”. Theexpression of a gene may be increased 1.5 times or more, 2 times ormore, or 3 times or more, as compared with that observed in anon-modified strain. Furthermore, the phrase that “the expression of agene is increased” can include not only when the expression amount of atarget gene is increased in a strain that inherently expresses thetarget gene, but also when the gene is introduced into a strain thatdoes not inherently express the target gene, and expressed therein. Thatis, the phrase “the expression of a gene is increased” also can mean,for example, that the target gene is introduced into a strain that doesnot have the gene, and expressed therein.

The expression of a gene can be increased by, for example, increasingthe copy number of the gene.

The copy number of a gene can be increased by introducing the gene intothe chromosome of a host microorganism. A gene can be introduced into achromosome by, for example, using homologous recombination (Miller, J.H., Experiments in Molecular Genetics, 1972, Cold Spring HarborLaboratory). Only one copy, or two or more copies of a gene may beintroduced. For example, by performing homologous recombination using asequence which is present in multiple copies on a chromosome as atarget, multiple copies of a gene can be introduced into the chromosome.Examples of such a sequence which is present in multiple copies on achromosome include repetitive DNAs, and inverted repeats located at theboth ends of a transposon. Alternatively, homologous recombination maybe performed by using an appropriate sequence on a chromosome such as agene unnecessary for production of the objective substance as a target.Homologous recombination can be performed by, for example, a method ofusing a linear DNA such as Red-driven integration (Datsenko, K. A., andWanner, B. L., Proc. Natl. Acad. Sci. USA, 97:6640-6645 (2000)), amethod of using a plasmid having a temperature sensitive replicationorigin, a method of using a plasmid capable of conjugative transfer, amethod of using a suicide vector not having a replication origin thatfunctions in a host, or a transduction method using a phage.Furthermore, a gene can also be randomly introduced into a chromosome byusing a transposon or Mini-Mu (Japanese Patent Laid-open (Kokai) No.2-109985, U.S. Pat. No. 5,882,888, EP 805867 B1).

Introduction of a target gene into a chromosome can be confirmed bySouthern hybridization using a probe having a sequence complementary tothe whole gene or a part thereof, PCR using primers prepared on thebasis of the sequence of the gene, or the like.

Furthermore, the copy number of a gene can also be increased byintroducing a vector containing the target gene into a hostmicroorganism. For example, the copy number of a target gene can beincreased by ligating a DNA fragment containing the target gene with avector that functions in a host microorganism to construct an expressionvector of the gene, and transforming the host microorganism with theexpression vector. The DNA fragment including the target gene can beobtained by, for example, PCR using the genomic DNA of a microorganismhaving the target gene as the template. As the vector, a vectorautonomously replicable in the cell of the host microorganism can beused. The vector is preferably a multi-copy vector. Furthermore, thevector preferably has a marker such as an antibiotic resistance gene forselection of transformant. Furthermore, the vector may have a promoterand/or terminator for expressing the introduced gene. The vector may be,for example, a vector derived from a bacterial plasmid, a vector derivedfrom a yeast plasmid, a vector derived from a bacteriophage, cosmid,phagemid, or the like. Specific examples of vector autonomouslyreplicable in bacteria belonging to the family Enterobacteriaceae suchas Escherichia coli include, for example, pUC19, pUC18, pHSG299,pHSG399, pHSG398, pACYC184, pBR322, pSTV29 (all of these are availablefrom Takara Bio), pMW219 (NIPPON GENE), pTrc99A (Pharmacia), pPROKseries vectors (Clontech), pKK233-2 (Clontech), pET series vectors(Novagen), pQE series vectors (QIAGEN), pACYC series vectors, and broadhost-range vector RSF1010. Specific examples of vector autonomouslyreplicable in coryneform bacteria include pHM1519 (Agric. Biol. Chem.,48, 2901-2903 (1984)); pAM330 (Agric. Biol. Chem., 48, 2901-2903(1984)); plasmids obtained by improving these and having a drugresistance gene; plasmid pCRY30 described in Japanese Patent Laid-open(Kokai) No. 3-210184; plasmids pCRY21, pCRY2KE, pCRY2KX, pCRY31,pCRY3KE, and pCRY3KX described in Japanese Patent Laid-open (Kokai) No.2-72876 and U.S. Pat. No. 5,185,262; plasmids pCRY2 and pCRY3 describedin Japanese Patent Laid-open (Kokai) No. 1-191686; pAJ655, pAJ611, andpAJ1844 described in Japanese Patent Laid-open (Kokai) No. 58-192900;pCG1 described in Japanese Patent Laid-open (Kokai) No. 57-134500; pCG2described in Japanese Patent Laid-open (Kokai) No. 58-35197; and pCG4and pCG11 described in Japanese Patent Laid-open (Kokai) No. 57-183799.

When a gene is introduced, it is sufficient that the gene is able to beexpressed by the microorganism of the present invention. Specifically,it is sufficient that the gene is introduced so that it is expressedunder control of a promoter sequence that functions in the microorganismof the present invention. The promoter may be a promoter derived fromthe host, or a heterogenous promoter. The promoter may be the nativepromoter of the gene to be introduced, or a promoter of another gene. Asthe promoter, for example, a stronger promoter as described herein mayalso be used.

A terminator for termination of gene transcription may be locateddownstream of the gene. The terminator is not particularly limited solong as it functions in the microorganism of the present invention. Theterminator may be a terminator derived from the host, or a heterogenousterminator. The terminator may be the native terminator of the gene tobe introduced, or a terminator of another gene. Specific examples ofterminator include, for example, T7 terminator, T4 terminator, fd phageterminator, tet terminator, and trpA terminator.

Vectors, promoters, and terminators available in various microorganismsare disclosed in detail in “Fundamental Microbiology Vol. 8, GeneticEngineering, KYORITSU SHUPPAN CO., LTD, 1987”, and those can be used.

Furthermore, when two or more of genes are introduced, it is sufficientthat the genes each are able to be expressed by the microorganism of thepresent invention. For example, all the genes may be located on a singleexpression vector or a chromosome. Furthermore, the genes may beseparately located on two or more expression vectors, or separatelylocated on a single or two or more expression vectors and a chromosome.An operon made up of two or more genes may also be introduced.

The gene to be introduced is not particularly limited so long as itcodes for a protein that functions in the host. The gene to beintroduced may be a gene derived from the host, or may be a heterogenousgene. The gene to be introduced can be obtained by, for example, PCRusing primers designed on the basis of the nucleotide sequence of thegene and the genomic DNA of an organism having the gene or a plasmidcarrying the gene as a template. The gene to be introduced may also betotally synthesized, for example, on the basis of the nucleotidesequence of the gene (Gene, 60(1), 115-127 (1987)).

In addition, when a protein functions as a complex consisting of aplurality of subunits, a part or all of the plurality of subunits may bemodified, so long as the activity of the protein is eventuallyincreased. That is, for example, when the activity of a protein isincreased by increasing the expression of a gene, the expression of apart or all of the genes that code for the subunits may be enhanced. Itis usually preferable to enhance the expression of all of of the genescoding for the subunits. That is, for example, when the malatethiokinase activity is increased by increasing the expression of amalate thiokinase gene, the expression of either the mtkA or mtkB genemay be enhanced or both may be enhanced, which may be preferable. Also,for example, when the succinyl-CoA synthase activity is increased byincreasing the expression of a succinyl-CoA synthase gene, theexpression of either the sucC or sucD gene may be enhanced, or both maybe enhanced, and which may be preferable. Also, for example, when thesuccinyl-CoA:malate-CoA-transferase activity is increased by increasingthe expression of a succinyl-CoA:malate-CoA-transferase gene, theexpression of either the smtA or smtB gene may be enhanced or both maybe enhanced, which is preferable. Furthermore, the subunits constitutingthe complex may be derived from a single kind of organism or two or morekinds of organisms, so long as the complex has a function of theobjective protein. That is, for example, genes of the same organismcoding for a plurality of subunits may be introduced into a host, orgenes of different organisms coding for a plurality of subunits may beintroduced into a host.

Furthermore, the expression of a gene can be increased by improving thetranscription efficiency of the gene. The transcription efficiency of agene can be improved by, for example, replacing the promoter of the geneon a chromosome with a stronger promoter. The “stronger promoter” meansa promoter providing an improved transcription of a gene compared withan inherent wild-type promoter of the gene. Examples of strongerpromoters include, for example, the known high expression promoters suchas T7 promoter, trp promoter, lac promoter, tac promoter, thr promoter,trc promoter, tet promoter, araBAD promoter, rpoH promoter, PR promoter,and PL promoter. Examples of stronger promoters usable in coryneformbacteria include the artificially modified P54-6 promoter (Appl.Microbiol. Biotechnolo., 53, 674-679 (2000)), pta, aceA, aceB, adh, andamyE promoters inducible in coryneform bacteria with acetic acid,ethanol, pyruvic acid, or the like, and cspB, SOD, and tuf promoters,which are potent promoters capable of providing a large expressionamount in coryneform bacteria (Journal of Biotechnology, 104 (2003)311-323; Appl. Environ. Microbiol., 2005 December; 71 (12):8587-96).Furthermore, as the stronger promoter, a highly-active type of anexisting promoter may also be obtained by using various reporter genes.For example, by making the −35 and −10 regions in a promoter regioncloser to the consensus sequence, the activity of the promoter can beenhanced (WO00/18935). Examples of highly active-type promoter includevarious tac-like promoters (Katashkina J I et al., Russian FederationPatent Application No. 2006134574) and pnlp 8 promoter (WO2010/027045).Methods for evaluating the strength of promoters and examples of strongpromoters are described in the paper of Goldstein et al. (ProkaryoticPromoters in Biotechnology, Biotechnol. Annu. Rev., 1, 105-128 (1995)),and so forth.

Furthermore, the expression of a gene can also be increased by improvingthe translation efficiency of the gene. The translation efficiency of agene can be improved by, for example, replacing the Shine-Dalgarno (SD)sequence (also referred to as ribosome binding site (RBS)) for the geneon a chromosome with a stronger SD sequence. The “stronger SD sequence”means a SD sequence that provides an improved translation of mRNAcompared with the inherentwild-type SD sequence of the gene. Examples ofstronger SD sequences include, for example, RBS of the gene 10 derivedfrom phage T7 (Olins P. O. et al, Gene, 1988, 73, 227-235). Furthermore,it is known that substitution, insertion, or deletion of severalnucleotides in a spacer region between RBS and the start codon,especially in a sequence immediately upstream of the start codon(5′-UTR), significantly affects the stability and translation efficiencyof mRNA, and hence, the translation efficiency of a gene can also beimproved by modifying them.

Sites that affect the gene expression, such as a promoter, SD sequence,and spacer region between RBS and the start codon, are also collectivelycalled “expression control regions”. An expression control region can beidentified by using a promoter search vector or gene analysis softwaresuch as GENETYX. Such an expression control region can be modified by,for example, a method of using a temperature sensitive vector or the Reddriven integration method (WO2005/010175).

The translation efficiency of a gene can also be improved by, forexample, modifying codons. In Escherichia coli etc., a clear codon biasexists among the 61 amino acid codons found within the population ofmRNA molecules, and the level of cognate tRNA appears directlyproportional to the frequency of codon usage (Kane, J. F., Curr. Opin.Biotechnol., 6 (5), 494-500 (1995)). That is, if there is a large amountof mRNA containing an excess amount of rare codons, a translationalproblem may arise. According to the recent research, it is suggestedthat clusters of AGG/AGA, CUA, AUA, CGA, or CCC codons may especiallyreduce both the quantity and quality of a synthesized protein. Such aproblem occurs especially when expressing a heterologous gene.Therefore, in the case of heterogenous expression of a gene or the like,the translation efficiency of the gene can be improved by replacing arare codon present in the gene with a synonymous codon more frequentlyused. Codons can be replaced by, for example, the site-specific mutationmethod for introducing an objective mutation into an objective site ofDNA. Examples of the site-specific mutation method include the methodutilizing PCR (Higuchi, R., 61, in PCR Technology, Erlich, H. A. Eds.,Stockton Press (1989); Carter, P., Meth. in Enzymol., 154, 382 (1987)),and the method utilizing phage (Kramer, W. and Frits, H. J., Meth. inEnzymol., 154, 350 (1987); Kunkel, T. A. et al., Meth. in Enzymol., 154,367 (1987)). Alternatively, a gene fragment in which objective codonsare replaced may be totally synthesized. Frequencies of codons invarious organisms are disclosed in the “Codon Usage Database”(http://www.kazusa.or.jp/codon; Nakamura, Y. et al, Nucl. Acids Res.,28, 292 (2000)).

Furthermore, the expression of a gene can also be increased byamplifying a regulator that increases the expression of the gene, ordeleting or attenuating a regulator that reduces the expression of thegene.

Such methods for increasing the gene expression as mentioned above maybe used independently or in an arbitrary combination.

Furthermore, a modification that increases the activity of a protein canalso be attained by, for example, enhancing the specific activity of theenzyme. Enhancement of the specific activity also includes reduction orelimination of feedback inhibition. A protein showing an enhancedspecific activity can be obtained by, for example, searching variousorganisms. Furthermore, a highly-active type of a native protein mayalso be obtained by introducing a mutation into the native protein. Themutation to be introduced may be, for example, substitution, deletion,insertion, or addition of one or several amino acid residues at one orseveral position of the protein. The mutation can be introduced by, forexample, such a site-specific mutation method as mentioned above. Themutation may also be introduced by, for example, a mutagenesistreatment. Examples of the mutagenesis treatment include irradiation ofX-ray, irradiation of ultraviolet, and a treatment with a mutation agentsuch as N-methyl-N′-nitro-N-nitrosoguanidine (MNNG), ethylmethanesulfonate (EMS), and methyl methanesulfonate (MMS). Furthermore,a random mutation may be induced by directly treating DNA in vitro withhydroxylamine. Enhancement of the specific activity may be independentlyused, or may be used in an arbitrary combination with such methods forenhancing gene expression as mentioned above.

The method for the transformation is not particularly limited, andconventionally known methods can be used. There can be used, forexample, a method of treating recipient cells with calcium chloride soas to increase the permeability thereof for DNA, which has been reportedfor the Escherichia coli K-12 strain (Mandel, M. and Higa, A., J. Mol.Biol., 1970, 53, 159-162), and a method of preparing competent cellsfrom cells which are in the growth phase, followed by transformationwith DNA, which has been reported for Bacillus subtilis (Duncan, C. H.,Wilson, G. A. and Young, F. E., Gene, 1977, 1:153-167). Alternatively,there can also be used a method of making DNA-recipient cells intoprotoplasts or spheroplasts, which can easily take up recombinant DNA,followed by introducing a recombinant DNA into the DNA-recipient cells,which is known to be applicable to Bacillus subtilis, actinomycetes, andyeasts (Chang, S. and Choen, S. N., 1979, Mol. Gen. Genet., 168:111-115;Bibb, M. J., Ward, J. M. and Hopwood, O. A., 1978, Nature, 274:398-400;Hinnen, A., Hicks, J. B. and Fink, G R., 1978, Proc. Natl. Acad. Sci.USA, 75:1929-1933). Furthermore, the electric pulse method reported forcoryneform bacteria (Japanese Patent Laid-open (Kokai) No. 2-207791) canalso be used.

An increase in the activity of a protein can be confirmed by measuringthe activity of the protein.

An increase in the activity of a protein can also be confirmed byconfirming an increase in the expression of a gene coding for theprotein. An increase in the expression of a gene can be confirmed byconfirming an increase in the transcription amount of the gene, or byconfirming an increase in the amount of a protein expressed from thegene.

An increase of the transcription amount of a gene can be confirmed bycomparing the amount of mRNA transcribed from the gene with thatobserved in a non-modified strain such as a wild-type strain or parentstrain. Examples of the method for evaluating the amount of mRNA includeNorthern hybridization, RT-PCR, and so forth (Sambrook, J., et al.,Molecular Cloning A Laboratory Manual/Third Edition, Cold spring HarborLaboratory Press, Cold Spring Harbor (USA), 2001). The amount of mRNAmay increase, for example, 1.5 times or more, 2 times or more, or 3times or more, as compared with that of a non-modified strain.

An increase in the amount of a protein can be confirmed by Westernblotting using antibodies (Molecular Cloning, Cold Spring HarborLaboratory Press, Cold Spring Harbor (USA), 2001). The amount of theprotein may increase, for example, 1.5 times or more, 2 times or more,or 3 times or more, as compared with that of a non-modified strain.

The aforementioned methods for increasing the activity of a protein canbe applied to enhancement of the activity of an arbitrary protein suchas α-ketoglutarate synthase, and enhancement of the expression of anarbitrary gene such as genes coding for the those arbitrary proteins.

<1-5> Method for Reducing Activity of Protein

Hereafter, methods for reducing the activity of a protein will beexplained.

The expression “the activity of a protein is reduced” can mean that theactivity of the protein per cell is decreased as compared with that of anon-modified strain such as a wild-type strain or parent strain, andincludes a state that the activity has completely disappeared.Specifically, the expression “the activity of a protein is reduced” canmean that the number of molecules of the protein per cell is reduced,and/or the function of each molecule of the protein is reduced ascompared with those of a non-modified strain. That is, the term“activity” in the expression “the activity of a protein is reduced” isnot limited to the catalytic activity of the protein, but may mean thetranscription amount of a gene (the amount of mRNA) coding for theprotein or the translation amount of the protein (the amount of theprotein). The phrase that “the number of molecules of the protein percell is reduced” can also include the absence of any protein. The phrasethat “the function of each molecule of the protein is reduced” caninclude when the function of each protein molecule completelydisappears. Although the degree of the reduction in the activity of aprotein is not particularly limited so long as the activity is reducedas compared with that of a non-modified strain, it may be reduced to,for example, 50% or less, 20% or less, 10% or less, 5% or less, or 0% ofthat of a non-modified strain.

The modification for reducing the activity of a protein can be attainedby, for example, reducing the expression of a gene coding for theprotein. The phrase that “the expression of a gene is reduced” caninclude when the gene is not expressed at all. The phrase that “theexpression of a gene is reduced” can also be referred to as “theexpression of a gene is attenuated”. The expression of a gene may bereduced to 50% or less, 20% or less, 10% or less, 5% or less, or 0% ofthat of a non-modified strain.

The reduction in gene expression may be due to, for example, a reductionin the transcription efficiency, a reduction in the translationefficiency, or a combination of them. The expression of a gene can bereduced by modifying an expression control sequence of the gene such asa promoter and Shine-Dalgarno (SD) sequence. When an expression controlsequence is modified, one or more nucleotides, two or more nucleotides,or three or more nucleotides, of the expression control sequence can bemodified. Furthermore, a part of or the entire expression controlsequence may be deleted. The expression of a gene can also be reducedby, for example, manipulating a factor responsible for expressioncontrol. Examples of the factor responsible for expression controlinclude low molecules responsible for transcription or translationcontrol (inducers, inhibitors, etc.), proteins responsible fortranscription or translation control (transcription factors etc.),nucleic acids responsible for transcription or translation control(siRNA etc.), and so forth.

The modification for reducing the activity of a protein can also beattained by, for example, disrupting a gene coding for the protein.Disruption of a gene can be attained by, for example, deleting a part ofor the entire coding region of the gene on a chromosome. Furthermore,entire genes including sequences upstream and downstream from the geneon a chromosome may be deleted. The region to be deleted may be anyregion such as an N-terminus region, an internal region, or a C-terminusregion, so long as the activity of the protein can be reduced. Deletionof a longer region will usually more surely inactivate the gene.Furthermore, it is preferred that reading frames of the sequencesupstream and downstream from the region to be deleted are not the same.

Disruption of a gene can also be attained by, for example, introducing amutation for an amino acid substitution (missense mutation), a stopcodon (nonsense mutation), a frame shift mutation which adds or deletesone or two nucleotide residues, or the like into the coding region ofthe gene on a chromosome (Journal of Biological Chemistry, 272:8611-8617(1997); Proceedings of the National Academy of Sciences, USA, 955511-5515 (1998); Journal of Biological Chemistry, 26 116, 20833-20839(1991)).

Disruption of a gene can also be attained by, for example, insertinganother sequence into a coding region of the gene on a chromosome. Siteof the insertion may be in any region of the gene, and insertion of alonger region will usually more surely inactivate the gene. It ispreferred that reading frames of the sequences upstream and downstreamfrom the insertion site are not the same. The other sequence is notparticularly limited so long as a sequence that reduces or eliminatesthe activity of the encoded protein is chosen, and examples thereofinclude, for example, a marker gene such as antibiotic resistance genes,and a gene useful for production of an objective substance.

Such modification of a gene on a chromosome as described above can beattained by, for example, preparing a deficient-type gene in which apartial sequence of the gene is deleted so that it cannot produce aprotein that can normally function, and transforming a microorganismwith a recombinant DNA including the deficient-type gene to causehomologous recombination between the deficient-type gene and thewild-type gene on a chromosome and thereby substitute the deficient-typegene for the wild-type gene on the chromosome. In this procedure, if amarker gene selected according to the characteristics of the host suchas auxotrophy is included in the recombinant DNA, the operation becomeseasy. The protein encoded by the deficient-type gene has a conformationdifferent from that of the wild-type protein, even if it is produced,and thus the function thereof is reduced or eliminated. Such genedisruption based on gene substitution utilizing homologous recombinationhas already been established, and there are methods of using a linearDNA such as a method called “Red driven integration” (Datsenko, K. A,and Wanner, B. L., Proc. Natl. Acad. Sci. USA, 97:6640-6645 (2000)), anda method utilizing the Red driven integration in combination with anexcision system derived from λ phage (Cho, E. H., Gumport, R. I.,Gardner, J. F., J. Bacteriol., 184:5200-5203 (2002)) (refer toWO2005/010175), a method of using a plasmid including a temperaturesensitive replication origin, a method of using a plasmid capable ofconjugative transfer, a method of utilizing a suicide vector notincluding a replication origin that functions in a host (U.S. Pat. No.6,303,383, Japanese Patent Laid-open (Kokai) No. 05-007491), and soforth.

The modification for reducing the activity of a protein can also beattained by, for example, a mutagenesis treatment. Examples of themutagenesis treatment include irradiation of X-ray, irradiation ofultraviolet, and treatment with a mutation agent such asN-methyl-N′-nitro-N-nitrosoguanidine (MNNG), ethyl methanesulfonate(EMS), and methyl methanesulfonate (MMS).

When a protein functions as a complex having a plurality of subunits,some or all of the subunits may be modified, so long as the activity ofthe protein is eventually reduced. That is, for example, some or all ofthe genes that code for the respective subunits may be disrupted or thelike. Furthermore, when there is a plurality of isozymes of a protein, apart or all of the activities of the plurality of isozymes may bereduced, so long as the activity of the protein is eventually reduced.That is, for example, some or all of of the genes that code for therespective isozymes may be disrupted or the like.

A reduction in the activity of a protein can be confirmed by measuringthe activity of the protein.

A reduction in the activity of a protein can also be confirmed byconfirming a reduction in the expression of a gene encoding the protein.A reduction in the expression of a gene can be confirmed by confirming areduction in the transcription amount of the gene or a reduction in theamount of the protein expressed from the gene.

A reduction in the transcription amount of a gene can be confirmed bycomparing the amount of mRNA transcribed from the gene with thatobserved in a non-modified strain. Examples of the method for evaluatingthe amount of mRNA include Northern hybridization, RT-PCR, and so forth(Molecular Cloning, Cold spring Harbor Laboratory Press, Cold SpringHarbor (USA), 2001). The amount of mRNA can be decreased to, forexample, 50% or less, 20% or less, 10% or less, 5% or less, or 0%, ofthat observed in a non-modified strain.

A reduction in the amount of a protein can be confirmed by Westernblotting using antibodies (Molecular Cloning, Cold Spring HarborLaboratory Press, Cold Spring Harbor (USA) 2001). The amount of theprotein can be decreased to, for example, 50% or less, 20% or less, 10%or less, 5% or less, or 0%, of that observed in a non-modified strain.

Disruption of a gene can be confirmed by determining nucleotide sequenceof a part or the whole of the gene, restriction enzyme map, full length,or the like of the gene depending on the means used for the disruption.

The aforementioned methods for reducing the activity of a protein can beapplied to reduction in the activity of an arbitrary protein such asdicarboxylic acid exporter proteins, and reduction in the expression ofan arbitrary gene such as genes coding for those arbitrary proteins.

<2> Method for Producing Objective Substance of the Present Invention

The methods of the present invention include a method for producing anobjective substance by culturing the microorganism of the presentinvention in a medium to produce and accumulate the objective substancein the medium or in cells of the microorganism, and collecting theobjective substance from the medium or the cells. One kind of objectivesubstance may be produced, or two or more kinds or objective substancesmay be produced.

The medium is not particularly limited, so long as the microorganism ofthe present invention can proliferate in the medium and produce anobjective substance. As the medium, for example, a typical medium usedfor culture of microorganisms such as bacteria can be used. As themedium, for example, a medium containing a carbon source, nitrogensource, phosphorus source, and sulfur source, as well as othercomponents such as various organic components and inorganic componentsas required can be used. The types and concentrations of the mediumcomponents can be appropriately determined according to variousconditions such as the type of the microorganism to be used and the typeof the objective substance to be produced.

The carbon source is not particularly limited, so long as themicroorganism of the present invention can utilize it and produce anobjective substance. Specific examples of the carbon source include, forexample, saccharides such as glucose, fructose, sucrose, lactose,galactose, xylose, arabinose, blackstrap molasses, hydrolysate ofstarches, and hydrolysate of biomass; organic acids such as acetic acid,fumaric acid, citric acid, succinic acid, and malic acid; alcohols suchas ethanol, glycerol, and crude glycerol; and fatty acids. As the carbonsource, plant-derived materials can be preferably used. Examples of theplant include, for example, corn, rice, wheat, soybean, sugarcane, beet,and cotton. Examples of the plant-derived materials include, forexample, organs such as root, stem, trunk, branch, leaf, flower, andseed, plant bodies including them, and decomposition products of theseplant organs. The forms of the plant-derived materials at the time ofuse thereof are not particularly limited, and they can be used in anyform such as unprocessed product, juice, ground product, and purifiedproduct. Pentoses such as xylose, hexoses such as glucose, or mixturesof them can be obtained from, for example, plant biomass, and used.Specifically, these saccharides can be obtained by subjecting a plantbiomass to such a treatment as steam treatment, hydrolysis withconcentrated acid, hydrolysis with diluted acid, hydrolysis with anenzyme such as cellulase, and alkaline treatment. Since hemicellulose isgenerally more easily hydrolyzed compared with cellulose, hemicellulosein a plant biomass may be hydrolyzed beforehand to liberate pentoses,and then cellulose may be hydrolyzed to generate hexoses. Furthermore,xylose may be supplied by conversion from hexoses by, for example,imparting a pathway for converting hexose such as glucose to xylose tothe microorganism of the present invention. As the carbon source, onekind of carbon source may be used, or two or more kinds of carbonsources may be used in combination.

The concentration of the carbon source in the medium is not particularlylimited, so long as the microorganism of the present invention canproliferate and produce an objective substance. It is preferable to makethe concentration of the carbon source in the medium as high as possiblewithin such a range that production of the objective substance is notinhibited. Initial concentration of the carbon source in the medium maybe, for example, usually 5 to 30% (w/v), preferably 10 to 20% (w/v).Furthermore, in accordance with consumption of the carbon sourceaccompanying progress of the fermentation, the carbon source may beadditionally added.

Specific examples of the nitrogen source include, for example, ammoniumsalts such as ammonium sulfate, ammonium chloride, and ammoniumphosphate, organic nitrogen sources such as peptone, yeast extract, meatextract, and soybean protein decomposition product, ammonia, and urea.Ammonia gas and aqueous ammonia used for pH adjustment may also be usedas a nitrogen source. As the nitrogen source, one kind of nitrogensource may be used, or two or more kinds of nitrogen sources may be usedin combination.

Specific examples of the phosphate source include, for example,phosphate salts such as potassium dihydrogenphosphate and dipotassiumhydrogenphosphate, and phosphoric acid polymers such as pyrophosphoricacid. As the phosphate source, one kind of phosphate source may be used,or two or more kinds of phosphate sources may be used in combination.

Specific examples of the sulfur source include, for example, inorganicsulfur compounds such as sulfates, thiosulfates, and sulfites, andsulfur-containing amino acids such as cysteine, cystine, andglutathione. As the sulfur source, one kind of sulfur source may beused, or two or more kinds of sulfur sources may be used in combination.

Specific examples of other various organic and inorganic componentsinclude, for example, inorganic salts such as sodium chloride andpotassium chloride; trace metals such as iron, manganese, magnesium andcalcium; vitamins such as vitamin B1, vitamin B2, vitamin B6, nicotinicacid, nicotinamide, and vitamin B12; amino acids; nucleic acids; andorganic components containing these such as peptone, casamino acid,yeast extract, and soybean protein decomposition product. As the othervarious organic and inorganic components, one kind of component may beused, or two or more kinds of components may be used in combination.

Furthermore, when an auxotrophic mutant strain that requires an aminoacid or the like for growth thereof is used, to the medium can besupplemented with the required nutrient. Furthermore, when L-glutamicacid is produced by using a coryneform bacterium, it is preferable to,for example, restrict the amount of biotin in the medium, or add asurfactant or penicillin to the medium. Furthermore, in order to preventfoaming during the culture, it is preferable to add an appropriateamount of a commercially-available antifoaming agent to the medium.

Culture conditions are not particularly limited, so long as themicroorganism of the present invention can proliferate and produce anobjective substance. The culture can be performed with, for example,usual conditions used for culture of microorganisms such as bacteria.The culture conditions may be appropriately determined according tovarious conditions such as the type of the microorganism to be used andthe type of the objective substance to be produced.

The culture can be performed by using a liquid medium. At the time ofthe culture, the microorganism of the present invention cultured on asolid medium such as agar medium may be directly inoculated into aliquid medium, or the microorganism of the present invention cultured ina liquid medium as seed culture may be inoculated into a liquid mediumfor main culture. That is, the culture may be performed separately asseed culture and main culture. The amount of the microorganism of thepresent invention contained in the medium at the time of the start ofthe culture is not particularly limited. For example, seed cultureshowing an OD660 of 4 to 8 may be added to a medium for main culture ata ratio of 0.1 to 30 mass %, or 1 to 10 mass %, at the time of the startof the culture.

The culture can be performed as batch culture, fed-batch culture,continuous culture, or a combination of these. Furthermore, when theculture is performed separately as seed culture and main culture, theculture schemes of the seed culture and the main culture may be or maynot be the same. For example, both the seed culture and the main culturemay be performed as batch culture. Alternatively, for example, the seedculture may be performed as batch culture, and the main culture may beperformed as fed-batch culture or continuous culture.

The culture may be performed under an aerobic condition, microaerobiccondition, or anaerobic condition. The culture can be performed under amicroaerobic condition or anaerobic condition. The aerobic conditionmeans that dissolved oxygen concentration in the liquid medium is notlower than 0.33 ppm, which is the detection limit for the detection withan oxygen membrane electrode, preferably not lower than 1.5 ppm. Themicroaerobic condition means that, although oxygen is supplied to theculture system, dissolved oxygen concentration in the liquid medium islower than 0.33 ppm. The anaerobic condition means that oxygen is notsupplied to the culture system. The culture may be performed under thecondition chosen above during the entire culture period, or during onlya part of the culture period. That is, “to culture under an aerobiccondition” means that the culture is performed under an aerobiccondition during at least a part of the culture period. Furthermore, “toculture under a microaerobic condition” means that the culture isperformed under a microaerobic condition during at least a part of theculture period. Furthermore, “to culture under an anaerobic condition”means that the culture is performed under an anaerobic condition duringat least a part of the culture period. The “part of the culture period”may be, for example, a period of 50% or more, 70 or more, 80% or more,90% or more, 95% or more, or 99% or more, of the whole culture period.When the culture is performed separately as seed culture and mainculture, the “entire culture period” may mean the entire period of themain culture. Specifically, the culture under an aerobic condition canbe performed by aeration culture or shaking culture. The microaerobiccondition or anaerobic condition can be attained by means of reducingaeration volume or stirring velocity, performing the culture in a sealedvessel without aeration, aerating an inert gas containing carbon dioxidegas, or the like to reduce dissolved oxygen concentration in the liquidmedium.

The pH of the medium may be, for example, 3 to 10, or 4.0 to 9.5. The pHof the medium can be adjusted during the culture as required. The pH ofthe medium can be adjusted by using various alkaline and acidicsubstances such as ammonia gas, aqueous ammonia, sodium carbonate,sodium bicarbonate, potassium carbonate, potassium bicarbonate,magnesium carbonate, sodium hydroxide, calcium hydroxide, and magnesiumhydroxide.

The medium may contain carbonate ions, bicarbonate ions, carbon dioxidegas, or a combination of these. These components can be supplied, forexample, by metabolism of the microorganism of the present invention, orfrom carbonate salt and/or bicarbonate salt used for pH adjustment.These components may also be supplied from carbonic acid, bicarbonicacid, salts thereof, or carbon dioxide gas, as required. Specificexamples of salts of carbonic acid or bicarbonic acid include, forexample, calcium carbonate, magnesium carbonate, ammonium carbonate,sodium carbonate, potassium carbonate, ammonium bicarbonate, sodiumbicarbonate, and potassium bicarbonate. Carbonate ions and/orbicarbonate ions may be added at a concentration of 0.001 to 5 M, 0.1 to3 M, 1 to 2 M. When carbon dioxide gas is contained, carbon dioxide gasmay be contained in an amount of 50 mg to 25 g, 100 mg to 15 g, or 150mg to 10 g, per litter of the solution.

The culture temperature may be, for example, 20 to 45° C., or 25 to 37°C. The culture time may be, for example, 1 hour or longer, 4 hours orlonger, 10 hours or longer, or 15 hours or longer, and may be 168 hoursor shorter, 120 hours or shorter, 90 hours or shorter, or 72 hours orshorter. Specifically, the culture time may be, for example, 10 to 120hours. The culture may be continued, for example, until the carbonsource contained in the medium is consumed, or until the activity of themicroorganism of the present invention is lost.

By culturing the microorganism of the present invention under suchconditions as described above, an objective substance can be accumulatedin the medium or in cells of the microorganism.

Moreover, when L-glutamic acid is produced, the culture can be performedwhile precipitating L-glutamic acid in the medium by using a liquidmedium adjusted to satisfy a condition under which L-glutamic acidprecipitates. Examples of the condition under which L-glutamic acidprecipitates include, for example, pH of 5.0 to 3.0, pH 4.9 to 3.5, pH4.9 to 4.0, or about pH 4.7 (European Patent Laid-open No. 1078989). Thetotal period or a partial period of the culture may be performed at theaforementioned pH. The “partial period” may be a period such asexemplified above.

Production of an objective substance can be confirmed by known methodsused for detection or identification of compounds. Examples of suchmethods include, for example, HPLC, LC/MS, GC/MS, and NMR. These methodscan be independently used, or can be used in an appropriate combination.

The produced objective substance can be collected by known methods usedfor separation and purification of compounds. Examples of such methodsinclude, for example, ion-exchange resin method, membrane treatment,precipitation, and crystallization. These methods can be independentlyused, or can be used in an appropriate combination. When an objectivesubstance is accumulated in cells of the microorganism, the cells can bedisrupted with, for example, ultrasonic waves or the like, and then theobjective substance can be collected by the ion exchange resin method orthe like from supernatant obtained by removing the cells from thecell-disrupted suspension by centrifugation. The collected objectivesubstance may be a free compound, a salt thereof, or a mixture of them.That is, the term “objective substance” used in the present inventionmay mean an objective substance in the free form, a salt thereof, or amixture of them, unless otherwise stated. Examples of the salt include,for example, sulfate salt, hydrochloride salt, carbonate salt, ammoniumsalt, sodium salt, and potassium salt. For example, L-glutamic acid mayalso be L-glutamic acid in the free form, monosodium glutamate (MSG),monoammonium glutamate, or a mixture thereof. For example, in the caseof L-glutamic acid, monosodium L-glutamate (MSG) can be obtained bycrystalizing monoammonium L-glutamate in the fermentation broth byaddition of an acid, and then by adding an equimolar of sodium hydroxideto the crystal. In addition, decolorization can be performed by usingactivated carbon before and/or after the crystallization (see, TetsuyaKAWAKITA, “Industrial Crystallization for Monosodium L-Glutamate.”,Bulletin of the Society of Sea Water Science, Japan, Vol. 56:5).

Furthermore, when the objective substance precipitates into the medium,it can be collected by centrifugation or filtration. An objectivesubstance precipitated into the medium and an objective substancedissolved in the medium may be isolated together after the objectivesubstance dissolved in the medium is crystallized.

Moreover, in cases where an objective substance is volatile, theobjective substance may be volatilized and collected. For example, anobjective substance can be efficiently volatilized and separated from aculture broth by aeration culture. The method for collecting thevolatilized objective substance is not particularly limited. Forexample, the volatilized objective substance may be accommodated in acollecting member such as a sealed vessel for general use or may betrapped in a proper liquid. Specifically, for example, a method oftrapping isopropyl alcohol or acetone in a liquid to thereby collect itis disclosed in WO2009/008377. Moreover, examples of a machineappropriate in such a method include, for example, a culturing apparatusdescribed in FIG. 1 of WO2009/008377.

The collected objective substance may contain, for example, cells of themicroorganism, medium components, moisture, and by-product metabolitesof the microorganism, in addition to the objective substance. Purity ofthe collected objective substance may be, for example, 30% (w/w) orhigher, 50% (w/w) or higher, 70% (w/w) or higher, 80% (w/w) or higher,90% (w/w) or higher, or 95% (w/w) or higher.

When the objective substance is L-glutamic acid, for example, themonosodium L-glutamate crystal can be used as an umami seasoning. Themonosodium L-glutamate crystal can be used as a seasoning in combinationwith a nucleic acid such as 5′-GMP disodium salt and 5′-IMP disodiumsalt, which also have umami taste.

EXAMPLES

Hereafter, the present invention will be more specifically explainedwith reference to examples. However, the present invention is notlimited by these examples.

Example 1 Effects of Dicarboxylic Acid Exporter Protein Deficiency inGlutamate Production by E. coli (1)

In this Example, dicarboxylic acid exporter gene-deficient strainsderived from E. coli MG1655 (ATCC 47076) were constructed to produceglutamic acid.

<1-1> Construction of the MG1655 ΔsucA ΔgadA ΔgadB Strain

The gadA and gadB genes on the genome (chromosome) of the E. coli MG1655ΔsucA strain (WO2007/125954) were deleted to construct the MG1655 ΔsucAΔgadA ΔgadB strain. The gene deletion was performed with a combinationmethod of “Red-driven integration” (Proc. Natl. Acad. Sci. USA, 2000,vol. 97, No. 12, p 6640-6645) and the excision system originated fromlambda phage (J. Bacteriol. 2002 September; 184(18): 5200-3.Interactions between integrase and excisionase in the phage lambdaexcisive nucleoprotein complex. Cho E H, Gumport R I, Gardner J F)(hereinafter also referred to as “λ-Red method”; WO2005/010175).Experimental procedures will be shown below.

A PCR reaction was performed using H70 (SEQ ID NO: 181) and H71 (SEQ IDNO: 182) (each containing a partial sequence of the gadB gene and eitherthe attL sequence or the attR sequence) as primers andpMW118-attL-Cm-attR (WO2005/010175) as a template. The obtained DNAfragment was digested with DpnI restriction enzyme and introduced byelectroporation to the MG1655 ΔsucA strain containing the plasmid pKD46,which has an ability to replicate in a thermo-sensitive manner (Proc.Natl. Acad. Sci. USA, 2000, vol. 97, No. 12, p 6640-6645).Chloramphenicol-resistant recombinants were selected by culturing at 30°C. on LB-agar medium containing Amp (ampicillin; 50 mg/L) and Cm(chloramphenicol; 20 mg/L). A colony PCR was performed using H72 (SEQ IDNO: 183) and H73 (SEQ ID NO: 184) as primers to select a recombinantstrain carrying gadB::Cm. The selected strain was named MG1655 ΔsucAgadB::Cm.

Next, a PCR reaction was performed using H66 (SEQ ID NO: 185) and H67(SEQ ID NO: 186) (each containing a partial sequence of the gadA geneand either the attL sequence or the attR sequence) as primers andpMW118-attL-Km-attR as a template. The obtained DNA fragment wasdigested with DpnI restriction enzyme and introduced by electroporationto the MG1655 ΔsucA gadB::Cm strain containing pKD46.Kanamycin-resistant recombinants were selected by culturing at 37° C. onLB-agar medium containing 50 mg/L of kanamycin (Km) and 25 mg/L of Cm. Acolony PCR was performed using H68 (SEQ ID NO: 187) and H69 (SEQ ID NO:188) as primers to select a recombinant strain carrying gadA::Km. Theselected strain was named MG1655 ΔsucA gadA::Km gadB::Cm. An Amps strainwas selected by single-colony isolation at 37° C., and the pMW-int-xisplasmid (WO2007/037460) was introduced to the Amps strain byelectroporation, and strains resistant to Amp were selected at 30° C.,followed by single-colony isolation of the obtained strains at 42° C. toobtain a strain of interest which had lost the drug-resistance cassettesand the plasmid: the MG1655 ΔsucA ΔgadA ΔgadB strain.

<1-2> Construction of the MG1655 ΔsucA ΔgadA ΔgadB ΔiscR Δldh ΔpflDΔpflB strain

In the above-obtained MG1655 ΔsucA ΔgadA ΔgadB strain, the iscR, ldh,pflD, and pflB genes on the genome were further deleted to construct theMG1655 ΔsucA ΔgadA ΔgadB ΔiscR Δldh ΔpflD ΔpflB strain. This strain wasproduced by preparing P1 phage from strains deficient in the respectivegenes in the Keio collection (Baba, T., Ara, T., Hasegawa, M., Takai, Y,Okumura, Y, Baba, M., Datsenko, K. A., Tomita, M., Wanner, B. L., andMori, H. (2006) Construction of Escherichia coli K-12 in-frame,single-gene knockout mutants: the Keio collection. Mol Syst Biol 2: 20060008) and repeatedly performing P1 transformation to the correspondingstrain of interest, and subsequent deletion of a drug-resistance geneused as a selection marker.

Specifically, at first, P1 transduction was performed using the E. coliBW25113 iscR::Kmr strain [rrnB3 ΔlacZ4787 hsdR514 Δ(araBAD)567Δ(rhaBAD)568 rph-1 iscR::Kmr] in the Keio collection as a donor andMG1655 ΔsucA ΔgadA ΔgadB as a recipient to obtain MG1655 ΔsucA ΔgadAΔgadB ΔiscR::Kmr. Then, a plasmid carrying a FLP recombinase gene,pMAN-FLP, was introduced to this strain to delete the Kmr gene with amethod described in Datsenko (Proc. Natl. Acad. Sci. USA, 2000, vol. 97,No. 12, p 6640-6645) and obtain MG1655 ΔsucA ΔgadA ΔgadB ΔiscR.

The above-used pMAN-FLP was produced by the following procedures. Thatis, pCP20 (PNAS 97: 6640-6645 (2000)) was treated with SmaI and BamHIrestriction enzymes to obtain a 3.3 kb fragment containing theFRT-specific Flp recombinase gene. The obtained DNA fragment was clonedinto the SmaI and BamHI sites in the multi-cloning site of the pMAN997vector (J. Bacteriol. 2001, 183(22):6538) to construct pMAN-FLP.

The genes ldh, pflD, and pflB were also deleted sequentially byrepeating the similar operations to eventually obtain the MG1655 ΔsucAΔgadA ΔgadB ΔiscR Δldh ΔpflD ΔpflB strain.

<1-3> Construction of the MG1655 ΔsucA ΔgadA ΔgadB ΔiscR Δldh ΔpflDΔpflB Δicd Strain

MG1655 Δicd::Cm is produced by the λ-Red method. A PCR reaction isperformed using 51 and S2 (each containing a partial sequence of the icdgene and either the attL sequence or the attR sequence) as primers andpMW118-attL-Cm-attR as a template. The obtained DNA fragment is digestedwith DpnI and introduced by electroporation to the MG1655 straincontaining the plasmid pKD46, which has an ability to replicate in athermo-sensitive manner. Cm-resistant recombinants are selected byculturing at 30° C. on LB-agar medium containing 50 mg/L of Amp and 20mg/L of Cm. A colony PCR is performed using the S3 and S4 primers toselect a recombinant strain carrying icd::Cm. The selected strain isnamed MG1655 Δicd::Cm.

P1 transduction is performed using MG1655 Δicd::Cm as a donor and MG1655ΔsucA ΔgadA ΔgadB ΔiscR Δldh ΔpflD ΔpflB as a recipient to obtain MG1655ΔsucA ΔgadA ΔgadB ΔiscR Δldh ΔpflD ΔpflB Δicd::Cm. Then, the pMW-int-xisplasmid is introduced to this strain by electroporation, and strainsresistant to Amp are selected at 30° C., followed by single-colonyisolation of the obtained strains at 42° C. to obtain a strain ofinterest which had lost the drug-resistance cassette and the plasmid:the MG1655 ΔsucA ΔgadA ΔgadB ΔiscR Δldh ΔpflD ΔpflB Δicd strain. Thisstrain is named E7-39 strain.

<1-4> Construction of a ΔyeeA Strain, a ΔynfM Strain, a ΔyjjP Strain,and a ΔyjjB Strain

MG1655 ΔyeeA:: Cm was produced by the λ-Red method. A PCR reaction wasperformed using the primer DEco yeeA-Fw(GGCCGACAGATGAGTTATGAGCGCTTTTAATCTCATTACGGAGTTTCTGCTGAAGCCTGCTTTTTTATACTAAGTTGGCA; SEQ ID NO: 221) and the primer DEcoyeeA-Rv (TTATCCTTGCTGAATCGAAGCAGCAGCAAGATGATTCTGAAGTTCAGGAACGCTCAAGTTAGTATAAAAAAGCTGAACGA; SEQ ID NO: 222) (each containing a partialsequence of the yeeA gene and either the attL sequence or the attRsequence) and pMW118-attL-Cm-attR as a template. The obtained DNAfragment was digested with DpnI restriction enzyme and introduced byelectroporation to the MG1655 strain containing the plasmid pKD46, whichhas an ability to replicate in a thermo-sensitive manner. Cm-resistantrecombinants were selected by culturing at 30° C. on LB-agar mediumcontaining 50 mg/L of Amp and 20 mg/L of Cm. A colony PCR was performedusing the primer DEco yeeA-CF (ATTACACTGTTCCCGGTTTGTCCGTCGGAT; SEQ IDNO: 223) and the primer DEco yeeA-CR (ATAGCTGCCGCAGATGACAATGCTTTTATC;SEQ ID NO: 224) to select a recombinant strain carrying yeeA:: Cm. Theselected strain was named MG1655 ΔyeeA::Cm.

MG1655 ΔynfM::Cm was produced by the λ-Red method. A PCR reaction wasperformed using the primer DEco ynfM-Fw(CTACCCTATGTATAAGCCTGATCTACAGGCATATTTAGCAAGGATTTCAATGAAGCCTGCTTTTTTATACTAAGTTGGCA; SEQ ID NO: 225) and the primer DEco ynfM-Rv(GAGCTGGCAATAAGTCCGGACGGGTATTTACCGCAGTCCGGACTTATTTTCGCTCAAGTTAGTATAAAAAAGCTGAACGA; SEQ ID NO: 226) (each containing a partialsequence of the ynfM gene and either the attL sequence or the attRsequence) and pMW118-attL-Cm-attR as a template. The obtained DNAfragment was digested with DpnI restriction enzyme and introduced byelectroporation to the MG1655 strain containing the plasmid pKD46, whichhas an ability to replicate in a thermo-sensitive manner. Cm-resistantrecombinants were selected by culturing at 30° C. on LB-agar mediumcontaining 50 mg/L of Amp and 20 mg/L of Cm. A colony PCR was performedusing the primer DEco ynfM-CF (AACATCTTATTTGAGATTATTAATATATTA; SEQ IDNO: 227) and the primer DEco ynfM-CR (GGAATTGGCTGGCGCTTCGTCTATTTTAGG;SEQ ID NO: 228) to select a recombinant strain carrying ynfM::Cm. Theselected strain was named MG1655 ΔynJM::Cm.

P1 transduction was performed using MG1655 ΔyeeA::Cm as a donor and theE7-39 strain as a recipient to obtain E7-39 ΔyeeA::Cm. Then, thepMW-int-xis plasmid was introduced to this strain by electroporation,and strains resistant to Amp were selected at 30° C., followed bysingle-colony isolation of the obtained strains at 42° C. to obtain astrain of interest which had lost the drug-resistance cassette and theplasmid: the E-39 ΔyeeA strain.

P1 transduction was performed using MG1655 ΔynJM::Cm as a donor and theE7-39 strain as a recipient to obtain E7-39 ΔynJM::Cm. Then, thepMW-int-xis plasmid was introduced to this strain by electroporation,and strains resistant to Amp were selected at 30° C., followed bysingle-colony isolation of the obtained strains at 42° C. to obtain astrain of interest which had lost the drug-resistance cassette and theplasmid: the E-39 ΔynfM strain.

P1 transduction was performed using MG1655 ΔynJM::Cm as a donor and theE7-39 ΔyeeA strain as a recipient to obtain E7-39 ΔyeeA ΔynJM::Cm. Then,the pMW-int-xis plasmid was introduced to this strain byelectroporation, and strains resistant to Amp were selected at 30° C.,followed by single-colony isolation of the obtained strains at 42° C. toobtain a strain of interest which had lost the drug-resistance cassetteand the plasmid: the E-39 ΔyeeA ΔynfM strain.

P1 transduction was performed using the BW25113 yjjP::Kmr strain [rrnB3ΔlacZ4787 hsdR514 Δ(araBAD)567 Δ(rhaBAD)568 rph-1 yjjP::Kmr] in the Keiocollection as a donor and E7-39 as a recipient to obtain E7-39ΔyjjP::Kmr. Then, a plasmid carrying a FLP recombinase gene, pMAN-FLP,was introduced to this strain to delete the Kmr gene with a methoddescribed in Datsenko (Proc. Natl. Acad. Sci. USA, 2000, vol. 97, No.12, p 6640-6645) and obtain E7-39 ΔyjjP.

P1 transduction was performed using the BW25113 yjjB::Kmr strain [rrnB3ΔlacZ4787 hsdR514 Δ(araBAD)567 Δ(rhaBAD)568 rph-1 yjjB::Kmr] in the Keiocollection as a donor and E7-39 as a recipient to obtain E7-39ΔyjjB::Kmr. Then, a plasmid carrying a FLP recombinase gene, pMAN-FLP,was introduced to this strain to delete the Kmr gene with a methoddescribed in Datsenko (Proc. Natl. Acad. Sci. USA, 2000, vol. 97, No.12, p 6640-6645) and obtain E7-39 ΔyjjB.

P1 transduction was performed using the BW25113 yjjP::Kmr strain [rrnB3ΔlacZ4787 hsdR514 Δ(araBAD)567 Δ(rhaBAD)568 rph-1 yjjP::Kmr] in the Keiocollection as a donor and E7-39 ΔyeeA ΔynfM as a recipient to obtainE7-39 ΔyeeA ΔynfM ΔyjjP::Kmr. Then, a plasmid carrying a FLP recombinasegene, pMAN-FLP, was introduced to this strain to delete the Kmr genewith a method described in Datsenko (Proc. Natl. Acad. Sci. USA, 2000,vol. 97, No. 12, p 6640-6645) and obtain E7-39 ΔyeeA ΔynfM ΔyjjP.

P1 transduction was performed using the BW25113 yjjB::Kmr strain [rrnB3ΔlacZ4787 hsdR514 Δ(araBAD)567 Δ(rhaBAD)568 rph-1 yjjB::Kmr] in the Keiocollection as a donor and E7-39 ΔyeeA ΔynfM as a recipient to obtainE7-39 ΔyeeA ΔynfM ΔyjjB::Kmr. Then, a plasmid carrying a FLP recombinasegene, pMAN-FLP, was introduced to this strain to delete the Kmr genewith a method described in Datsenko (Proc. Natl. Acad. Sci. USA, 2000,vol. 97, No. 12, p 6640-6645) and obtain E7-39 ΔyeeA ΔynfM ΔyjjB.

<1-5> Construction of the RSFPPG plasmid

RSFPPG (WO 2010027022 A1) is a plasmid obtained by replacing the gltAgene on RSFCPG (see EP0952221) with the prpC gene. RSFPPG was preparedby the following procedures.

The primers RSFBg1-2 (ggaagatctatttgccttcgcacatcaacctgg; SEQ ID NO: 209)and RSFKpn (cggggtaccttgtaaatallllaacccgcc; SEQ ID NO: 210) weredesigned to amplify the entire part of RSFCPG except for the ORF of thegltA gene. A PCR was performed using these primers and RSFCPG as atemplate to obtain a fragment of about 14.9 kb. Separately, for prpC, aPCR was performed using the primers coliprpCBgl-1(ggaagatctaaggagaccttaaatgagcgacacaacgatcctgcaaaacagtaccc; SEQ ID NO:211) and coliprpCKpn (cggggtacctcgtagaggtttactggcgcttatccagcg; SEQ IDNO: 212) and the genomic DNA of the E. coli strain W3110 as a templateto obtain a fragment of about 1.2 kb. Both the PCR products wereindependently treated with BglII and KpnI and ligated to each other, andthen used for transformation of the E. coli strain JM109. All of theformed colonies were collected and plasmids were extracted as a mixture.The ME8330 strain, which is deficient in gltA gene encoding a citratesynthase, was transformed with this plasmid mixture and applied onto anM9 minimal medium containing 50 mg/L of uracil and 5 mg/L ofthiamine-HCl. All of the formed colonies were collected and plasmidswere extracted as a mixture and the P. ananatis Glu-producing hostbacterial strain NP106 was transformed with this plasmid mixture. Astrain showing a yield comparable to that of the P. ananatis strain G106(AJ13601; FERM BP-7207) was named NA1. Furthermore, a plasmid wasextracted from this bacterial strain and this plasmid is named RSFPPG, aplasmid for expression of the prpC, gdh, and ppc genes.

<1-6> Cloning of the α-Ketoglutarate Synthase Gene, the PyruvateSynthase Gene, and the Ferredoxin Gene

A plasmid for expressing in E. coli the α-ketoglutarate synthase (KGS)gene, the pyruvate synthase (PS) gene, and the ferredoxin (Fd) genederived from Chlorobaculum tepidum was constructed. Experimentalprocedures will be shown below.

First, a PCR was performed according to an ordinary method and using thegenomic DNA of Chlorobaculum tepidum as a template and primers as shownin SEQ ID NO: 189 and SEQ ID NO: 190 to amplify the Fd gene. Moreover,the KGS gene was amplified in a similar way using SEQ ID NO: 191 and SEQID NO: 192 as primers. A PCR was performed using a mixture of theobtained Fd and KGS gene products as a template and SEQ ID NO: 189 andSEQ ID NO: 192 as primers to obtain a Fd-KGS gene fragment in which bothof the fragments were linked together. The purified Fd-KGS gene fragmentand a fragment of pMW219-Ptac-Ttrp (WO2013/069634), which had beentreated with SmaI restriction enzyme and subsequently purified, werelinked together by using the In-Fusion HD Cloning Kit (manufactured byClontech Laboratories, Inc.). The plasmid for expression of Fd-KGS geneswas named pMW219-Fd-KGS.

Next, a PCR was performed according to an ordinary method and using thegenomic DNA of Chlorobaculum tepidum as a template and primers as shownin SEQ ID NO: 193 and SEQ ID NO: 194 to amplify the PS gene. Moreover, aPCR was performed according to an ordinary method and using thepMW219-Fd-KGS plasmid as a template and primers as shown in SEQ ID NO:195 and SEQ ID NO: 196 to amplify the pMW219-Fd-KGS fragment. Theobtained two fragments were linked together by using the In-Fusion HDCloning Kit (manufactured by Clontech Laboratories, Inc.). The obtainedplasmid for expression of the Fd, KGS, and PS genes was namedpMW219-Fd-KGS-PS (also referred to as pMW219-FdKGSPS).

<1-7> Confirmation of Glutamate Production in Anaerobic Culture

Strains are produced by simultaneously introducing the RSFPPG plasmidand the pMW219-FdKGSPS plasmid to each of the strains E3-39, E3-39ΔyjjP, E3-39 ΔyjjB, E3-39 ΔyeeA, E3-39 ΔynfM, E3-39 ΔyeeA ΔynfMA, E3-39ΔyeeA ΔynfM ΔyjjP, and E3-39 ΔyeeA ΔynfM ΔyjjB.

Next, the L-glutamate production culture is performed by using theproduced bacterial strains to examine the ability to produce L-glutamicacid. The bacterial strains are uniformly applied onto LBM9Glc plates(produced by adding 200 mL of 5×M9 salts and 10 mL of 50% glucose to 800mL of LB medium) containing a suitable antibiotic (50 μg/mL of kanamycinor 15 μg/mL of tetracycline) and cultured at 37° C. for 16-20 hours.Then, those plates are placed into an AnaeroPack pouch (manufactured byMitsubishi Gas Chemical Company, Inc.; for easy cultivation of anaerobicbacteria; Product Number: A-04) and incubated under anaerobic conditionsat 37° C. for 6 hours. Obtained bacterial cells on the plates aresuspended in 700 μL of 0.8% saline to give an optical density (OD) of0.5 to 1.5 (600 nm) when diluted 51 times. To a screw cap microtubehaving a volume of 1.5 mL, 200 μl of this bacterial cell suspension and1 mL of the production medium purged with a sufficient amount of carbondioxide gas in advance (1 vvm, 30 minutes or longer) are placed andcovered tightly with the cap and then incubated using a microtube shakerunder anaerobic conditions at 37° C. for 24 or 48 hours. The compositionof the production medium is shown below.

The composition of the production medium

Part A: Glucose 10 g/L (final concentration) Part B: Magnesium sulfateheptahydrate 1 g/L Ammonium sulfate 15 g/L Monopotassium phosphate 1 g/LBiotin 1 mg/L Vitamin B1 1 mg/L Trace metal solution 10 mL/L (adjustedto pH = 7 with KOH) Part C: Calcium carbonate (Japanese Pharmacopeia) 50g/L

After Part A and Part B are separately sterilized by autoclaving at 115°C. for 10 minutes and Part C is sterilized by dry-heating at 180° C. for3 hours, they are left to cool and then mixed.

The trace metal solution is a solution containing, per liter, 1.35 g ofFeCl₂.6H₂O, 680 mg of ZnCl₂, 249 mg of CuSO₄.5H₂O, 120 mg of MnSO₄.5H₂O,118 mg of CoCl₂.6H₂O, and 123 mg of (NH₄)6Mo₇O₂₄.4H₂O.

After the culture, the concentrations of glutamic acid accumulated andthe sugar remaining in the medium are analyzed with the Biotech AnalyzerAS-310 (Sakura SI Co. Ltd.). Moreover, the amounts of other organicacids are analyzed with a liquid chromatography HPLC system (L-7100,L-7200, L-7300, or L-7400; Hitachi High-Technologies Co.) and theURUTRON PS-80H column (Shinwa Chemical Industries Ltd.). The turbidity(OD) of the bacterial cell suspension is measured using theSpectrophotometer DU800 (Beckman Coulter) after calcium carbonate in themedium is dissolved by diluting the sample with 0.1 N hydrochloric acid.

Example 2 Effects of Dicarboxylic Acid Exporter Protein Deficiency inGlutamate Production by E. aerogenes

In this Example, dicarboxylic acid exporter gene-deficient strainsderived from Enterobacter aerogenes AJ110637 (FERM BP-10955) wereconstructed to produce glutamic acid. AJ110637 (FERM BP-10955) has beendeposited in International Patent Organism Depositary, NationalInstitute of Advanced Industrial Science and Technology (currently,International Patent Organism Depositary, National Institute ofTechnology and Evaluation; address: #120, 2-5-8, Kazusakamatari,Kisarazu-shi, Chiba 292-0818 Japan) on Aug. 22, 2007 and has beenassigned the accession number FERM BP-10955.

<2-1> Construction of the ES06 Strain

The Enterobacter aerogenes ES06 strain was constructed by replacing thepoxB gene on the genome of the ES04 strain (US2010-0297716A1), which hadbeen constructed from the Enterobacter aerogenes strain AJ110637 (FERMBP-10955), with the pckA gene from the Actinobacillus succinogenesstrain 130Z. Experimental procedures will be shown below.

Construction of the λattL-Km^(r)-λattR-Ptac-pckA Gene Fragment

The entire nucleotide sequence of the genomic DNA of the Actinobacillussuccinogenes strain 130Z (ATCC 55618) has already been released (GenBankAccession No. CP000746) and a gene encoding a phosphoenolpyruvatecarboxykinase (gene name: pckA; accession number: Asuc_0221) has beenidentified. The nucleotide sequence of the pckA gene from theActinobacillus succinogenes strain 130Z is shown in SEQ ID NO: 213 andthe amino acid sequence of a phosphoenolpyruvate carboxykinase encodedby the same gene is shown in SEQ ID NO: 214. A PCR reaction (TaKaRaPrime star (registered trademark); 30 cycles of 94° C. for 10 sec., 54°C. for 20 sec., and 72° C. for 90 sec.) was performed using the genomicDNA of the Actinobacillus succinogenes strain 130Z as a template andprimers designed based on the above-described nucleotide sequence andlisted as SEQ ID NO: 215 and SEQ ID NO: 216 to obtain a DNA fragmentcontaining the pckA ORF region. Moreover, a PCR reaction (TaKaRa Primestar (registered trademark); 30 cycles of 94° C. for 10 sec., 54° C. for20 sec., and 72° C. for 90 sec.) was performed using a DNA fragmentcontaining λattL-Km^(r)λattR-Ptac (WO2008090770A1) as a template andprimers listed as SEQ ID NO: 217 and SEQ ID NO: 218 to obtain a DNAfragment containing λattL-Km^(r)λattR-Ptac. Then, a PCR reaction (TaKaRaPrime star (registered trademark); 35 cycles of 94° C. for 10 sec., 54°C. for 20 sec., and 72° C. for 180 sec.) was performed using the DNAfragment containing the pckA ORF region and the DNA fragment containingλattL-Km^(r)λattR-Ptac as templates and primers listed as SEQ ID NO: 216and SEQ ID NO: 217 to obtain the λattL-Km^(r)λattR-Ptac-pckA genefragment having sequences for recombination with a gene encoding apyruvate oxidase (gene name: poxB) at the respective ends.

Construction of the ES04/RSFRedTER Strain

The ES04 strain (US20100297716A1) was cultured overnight in liquid LBmedium. Then, 100 μL of the culture broth was inoculated into 4 mL offresh liquid LB medium and incubated with shaking at 34° C. for 3 hours.After bacterial cells were collected, they were washed three times with10% glycerol to make competent cells and introduced with RSFRedTER(WO2008/090770A1) by electroporation. The electroporation was carriedout by using the GENE PULSER II (manufactured by Bio-Rad Laboratories,Inc.) under the following conditions: electric field intensity, 20kV/cm; capacitance, 25 μf; electric resistance, 200Ω. After two hours ofculture in SOC medium (BACTO Tryptone, 20 g/L; yeast extract, 5 g/L;NaCl, 0.5 g/L; glucose, 10 g/L), the culture broth was applied onto LBmedium containing 40 mg/L of chloramphenicol and incubated for 16 hours.Eventually, a transformant exhibiting chloramphenicol resistance wasobtained and named ES04/RSFRedTER strain.

Construction of the ES04 ΔpoxB::λattL-Km^(r)λattR-Ptac-pckA Strain

The ES04/RSFRedTER strain was cultured overnight in liquid LB medium.Then, 1 mL of the culture broth was inoculated into 100 mL of liquid LBmedium containing final concentrations of 1 mM IPTG and 40 mg/Lchloramphenicol and incubated with shaking at 34° C. for 3 hours. Afterbacterial cells were collected, they were washed three times with 10%glycerol to make competent cells. The amplifiedλattL-Km^(r)-λattR-Ptac-pckA gene fragment was purified using the WizardPCR Prep DNA Purification System (manufactured by Promega Co.) andintroduced to the competent cells by electroporation. After two hours ofculture in SOC medium, the culture broth was applied onto LB mediumcontaining 50 mg/L of kanamycin and incubated for 16 hours. Afterpurifying the formed colonies with the same medium, a colony PCR (TaKaRaSpeed star (registered trademark); 40 cycles of 92° C. for 10 sec., 56°C. for 10 sec., and 72° C. for 30 sec.) was performed using primerslisted as SEQ ID NO: 219 and SEQ ID NO: 220 to confirm that the poxBgene on the genome was replaced with the λattL-Km^(r)-λattR-Ptac-pckAgene. The obtained strain was applied onto LB-agar medium containing 10%sucrose and 1 mM IPTG to remove the RSFRedTER plasmid and eventuallyobtain the ES04 ΔpoxB::λattL-Km^(r)-λattR-Ptac-pckA strain.

Removal of the Kanamycin-Resistance Gene from the ES04ΔpoxB::λattL-Km^(r)-λattR-Ptac-pckA Strain

The RSF-int-xis plasmid (US20100297716A1) was used to remove thekanamycin-resistance gene from the ES04ΔpoxB::λattL-Km^(r)-λattR-Ptac-pckA strain. The ES04ΔpoxB::λattL-Km^(r)-λattR-Ptac-pckA strain was introduced withRSF-int-xis by electroporation, applied onto LB medium containing 40mg/L of chloramphenicol, and cultured at 30° C. to obtain the ES04ΔpoxB::λattL-Km^(r)-λattR-Ptac-pckA/RSF-int-xis strain. The obtainedplasmid-carrying strain was purified with LB medium containing 40 mg/Lchloramphenicol and 1 mM IPTG to obtain plural single colonies. Then,the colonies were applied onto a medium supplemented with 50 mg/Lkanamycin and cultured overnight at 37° C. to confirm by identifying theinability of the colonies to grow that they were from a strain in whichthe kanamycin-resistance gene had been removed. Then, to remove theRSF-int-xis plasmid from the obtained strain, the strain was appliedonto LB medium supplemented with 10% sucrose and 1 mM IPTG and culturedovernight at 37° C. Among formed colonies, a strain exhibitingchloramphenicol sensitivity was named ES06 strain.

<2-2> Construction of the RSFPP Plasmid

The RSFPP plasmid was obtained by deleting a region containing the gdhAgene from RSFPPG (WO 2010027022 A1). Specifically, the RSFPPG plasmidwas treated with NspV restriction enzyme and treated by heating at 75°C. for 10 minutes to inactivate the enzyme and subsequently self-ligatedusing a DNA ligation kit manufactured by Takara Bio Inc. The E. colistrain DH5α was transformed with this DNA solution and selected onLB-agar medium containing 12.5 mg/L of tetracycline to obtain theDH5α/RSFPP strain.

<2-3> Construction of the ES06 ΔyeeA Strain

The ES06 ΔyeeA strain was produced by the λ-red method. Specifically, aPCR reaction was performed using primers as shown in SEQ ID NOs: 197 and198 and pMW118-attL-Kmr-attR (WO2008/090770A1) as a template to amplifya fragment having sequences of 50 bp complementary to the internalsequence of the yeeA gene in Enterobacter aerogenes at the respectiveends and containing the kanamycin-resistance gene flanked by the attLand attR sequences of λ phage. The ES06 strain carrying RSFRedTER (BMCMol. Biol. 10, 34 (2009)) was cultured overnight in liquid LB medium,and 1 mL of the culture broth was inoculated into 100 mL of liquid LBmedium containing final concentrations of 1 mM IPTG and 25 mg/Lchloramphenicol and incubated with shaking at 34° C. for 3 hours. Afterbacterial cells were collected, they were washed three times with 10%glycerol to make competent cells. The amplified PCR fragment waspurified using the Wizard PCR Prep manufactured by Promega Corporationand introduced to the competent cells by electroporation. Theelectroporation was carried out by using the GENE PULSER II(manufactured by Bio-Rad Laboratories, Inc.) under the followingconditions: electric field intensity, 20 kV/cm; capacitance, 25 μF;electric resistance, 200Ω. The ES06 ΔyeeA::Km strain was obtained byselection on LB-agar medium containing 40 mg/L of kanamycin. Theobtained strain was applied onto LB-agar medium containing M9 components(17.1 g/L Na₂HPO₄.12H₂O, 3 g/L KH₂PO₄, 0.5 g/L NaCl, 1 g/L NH₄Cl), 10%sucrose, and 1 mM IPTG to obtain a strain which had lost the RSFRedTERplasmid. This strain was introduced with the pMW-intxis-sacB plasmid(identical to pMW-intxis-sacB (Cm) plasmid disclosed in WO2015/005405)by electroporation and selected on LB-agar medium containing 25 mg/L ofchloramphenicol to obtain the ES06 ΔyeeA::Km/pMW-intxis-sacB strain.After purifying this strain on LB-agar medium, a replica was preparedwith LB-agar medium containing 40 mg/L of kanamycin to identify akanamycin-sensitive strain: the ES06 ΔyeeA strain.

<2-4> Construction of the ES06 ΔyeeA ΔynfM Strain

The ES06 ΔyeeA ΔynfM strain was produced from the ES06 ΔyeeA strain bythe above-described λ-red method. Specifically, a PCR reaction wasperformed using primers as shown in SEQ ID NOs: 199 and 200 andpMW118-attL-Kmr-attR as a template to amplify a fragment havingsequences of 50 bp complementary to the internal sequence of the ynfMgene in Enterobacter aerogenes at the respective ends and containing thekanamycin-resistance gene flanked by the attL and attR sequences of λphage. The ES06 ΔyeeA/RSFRedTER strain, which had been obtained byintroducing RSFRedTER into the ES06 ΔyeeA strain by electroporation, wascultured overnight in liquid LB medium, and 1 mL of the culture brothwas inoculated into 100 mL of liquid LB medium containing finalconcentrations of 1 mM IPTG and 25 mg/L chloramphenicol and incubatedwith shaking at 34° C. for 3 hours. After bacterial cells werecollected, they were washed three times with 10% glycerol to makecompetent cells. The amplified PCR fragment was purified using theWizard PCR Prep manufactured by Promega Corporation and introduced tothe competent cells by electroporation. The electroporation was carriedout by using the GENE PULSER II (manufactured by Bio-Rad Laboratories,Inc.) under the following conditions: electric field intensity, 20kV/cm; capacitance, 25 μf; electric resistance, 200Ω. The ES06 ΔyeeAΔynfM::Km strain was obtained by selection on LB-agar medium containing40 mg/L of kanamycin. The obtained strain was applied onto LB-agarmedium containing M9 components (17.1 g/L Na₂HPO₄.12H₂O, 3 g/L KH₂PO₄,0.5 g/L NaCl, 1 g/L NH₄Cl), 10% sucrose, and 1 mM IPTG to obtain astrain which had lost the RSFRedTER plasmid. This strain was introducedwith the pMW-intxis-sacB plasmid by electroporation and selected onLB-agar medium containing 25 mg/L of chloramphenicol to obtain the ES06ΔyeeA ΔynfM::Km/pMW-intxis-sacB strain. After purifying this strain onLB-agar medium, a replica was prepared with LB-agar medium containing 40mg/L of kanamycin to identify a kanamycin-sensitive strain: the ES06ΔyeeA ΔynfM strain.

<2-5> Construction of the sdhA-Disrupted Strain

The ES06 ΔsdhA strain, which is deficient in the sdhA gene encoding asubunit of a succinate dehydrogenase, was produced from the ES06 strainby the above-described λ-red method. Specifically, a PCR reaction wasperformed using primers as shown in SEQ ID NOs: 201 and 202 andpMW118-attL-Kmr-attR as a template to amplify a fragment havingsequences of 50 bp complementary to the internal sequence of the sdhAgene in Enterobacter aerogenes at the respective ends and containing thekanamycin-resistance gene flanked by the attL and attR sequences of λphage. The ES06 strain carrying RSFRedTER was cultured overnight inliquid LB medium, and 1 mL of the culture broth was inoculated into 100mL of liquid LB medium containing final concentrations of 1 mM IPTG and25 mg/L chloramphenicol and incubated with shaking at 34° C. for 3hours. After bacterial cells were collected, they were washed threetimes with 10% glycerol to make competent cells. The amplified PCRfragment was purified using the Wizard PCR Prep manufactured by PromegaCorporation and introduced to the competent cells by electroporation.The electroporation was carried out by using the GENE PULSER II(manufactured by Bio-Rad Laboratories, Inc.) under the followingconditions: electric field intensity, 20 kV/cm; capacitance, 25 μf;electric resistance, 200Ω. The ES06 ΔsdhA::Km strain was obtained byselection on LB-agar medium containing 40 mg/L kanamycin and 20 mMdisodium malate. The obtained strain was applied onto LB-agar mediumcontaining M9 components (17.1 g/L Na₂HPO₄.12H₂O, 3 g/L KH₂PO₄, 0.5 g/LNaCl, 1 g/L NH₄Cl), 10% sucrose, and 1 mM IPTG to obtain a strain whichhad lost the RSFRedTER plasmid. This strain was introduced with thepMW-intxis-sacB plasmid by electroporation and selected on LB-agarmedium containing 25 mg/L chloramphenicol and 20 mM disodium malate toobtain the ES06 ΔsdhA::Km/pMW-intxis-sacB strain. After purifying thisstrain on LB-agar medium containing 20 mM disodium malate, a replica wasprepared with LB-agar medium containing 40 mg/L kanamycin and 20 mMdisodium malate to identify a kanamycin-sensitive strain: the ES06 ΔsdhAstrain. Moreover, strains deficient in the sdhA gene were derivedsimilarly from the ES06 ΔyeeA strain and the ES06 ΔyeeA ΔynfM strain andnamed ES06 ΔsdhA ΔyeeA strain and ES06 ΔsdhA ΔyeeA ΔynfM strain,respectively. These strains were introduced with the RSFPP plasmid byelectroporation and selected on LB-agar medium containing 12.5 mg/Ltetracycline and 20 mM disodium malate to identify the respectivestrains: the ES06 ΔsdhA/RSFPP strain, the ES06 ΔsdhA ΔyeeA/RSFPP strain,and the ES06 ΔsdhA ΔyeeA ΔynfM/RSFPP strain.

<2-6> Glutamate Production

Next, the ability to produce glutamic acid was evaluated in thesestrains. The composition of the production medium is shown below.

The composition of the production medium

Part A: Sucrose 30 g/L MgSO₄•7H₂O 0.5 g/L Part B: (NH₄)₂SO₄ 2.0 g/LKH₂PO₄ 2.0 g/L Yeast Extract 2.0 g/L FeSO₄•7H₂O 0.02 g/L MnSO₄•5H₂O 0.02g/L L-lysine hydrochloride 0.2 g/L DL-methionine 0.2 g/L Diaminopimelicacid 0.2 g/L (adjusted to pH 7.0 with KOH) Part C: CaCO₃ 20 g/L

After Part A and Part B were separately sterilized by autoclaving at115° C. for 10 minutes and Part C is sterilized by dry-heating at 180°C. for 3 hours, they were mixed and tetracycline hydrochloride was addedthereto to 12.5 mg/L.

The ES06 ΔsdhA/RSFPP strain, the ES06 ΔsdhA ΔyeeA/RSFPP strain, and theES06 ΔsdhA ΔyeeA ΔynfM/RSFPP strain were cultured overnight at 34° C. onLBGM9 medium plates containing 12.5 mg/L of tetracycline and 15 g/L ofagar, and then an appropriate amount of the obtained bacterial cells ofeach strain was inoculated into a test tube containing 5 mL of theabove-described medium and incubated at 34° C. with shaking at 120 rpm.

The results are shown in Table 1. The ability to produce L-glutamic acidwas increased in the ES06 ΔsdhA ΔyeeA/RSFPP strain deficient in the yeeAgene and in the ES06 ΔsdhA ΔyeeA ΔynfM/RSFPP strain further deficient inthe ynfM gene as compared with the control ES06 ΔsdhA/RSFPP strain. Fromthe above results, it was revealed that the ability to produce glutamicacid is increased by deleting dicarboxylic acid exporter gene(s) inEnterobacter aerogenes.

TABLE 1 Concentration of accumulated glutamic Yield of glutamic acid ±acid ± S.E. (g/L) S.E. (%) ES-06 ΔsdhA/ 0.9 ± 0.0  2.6 ± 0.1 RSFPP ES-06ΔsdhA ΔyeeA/ 2.4 ± 0.1 10.0 ± 1.2 RSFPP ES-06 ΔsdhA ΔyeeA ΔynfM/ 3.0 ±0.1 17.0 ± 0.4 RSFPP

Example 3 Effects of Dicarboxylic Acid Exporter Protein Deficiency inGlutamate Production by P. ananatis

In this Example, dicarboxylic acid exporter gene-deficient strainsderived from the P. ananatis strain SC17(0) (VKPM B-9246) wereconstructed to produce glutamic acid. The SC17(0) strain has beendeposited in Russian National Collection of Industrial Microorganisms(VKPM), GNII Genetika (address: 1-st Dorozhny proezd, 1, 117545 Moscow,Russia) on Sep. 21, 2005 under the accession number VKPM B-9246.

<3-1> Construction of the P. ananatis yeeA Gene-Deficient Strain

A PCR reaction was performed using primers as shown in SEQ ID NOs: 203and 204 and pMW118-attL-Kmr-attR as a template to amplify a fragmenthaving sequences of 50 bp complementary to the internal sequence of theyeeA gene in Pantoea ananatis at the respective ends and containing thekanamycin-resistance gene flanked by the attL and attR sequences of λphage. The SC17(0) strain carrying RSFRedTER (BMC Molecular Biology2009, 10:34) was cultured overnight in liquid LB medium, and 1 mL of theculture broth was inoculated into 100 mL of liquid LB medium containingfinal concentrations of 1 mM IPTG and 25 mg/L chloramphenicol andincubated with shaking at 34° C. for 3 hours. After bacterial cells werecollected, they were washed three times with 10% glycerol to makecompetent cells. The amplified PCR fragment was purified using theWizard PCR Prep manufactured by Promega Corporation and introduced tothe competent cells by electroporation. The electroporation was carriedout by using the GENE PULSER II (manufactured by Bio-Rad Laboratories,Inc.) under the following conditions: electric field intensity, 20kV/cm; capacitance, 25 μF; electric resistance, 200Ω. The SC17(0)ΔyeeA::Km strain was obtained by selection on LB-agar medium containing40 mg/L of kanamycin.

<3-2> Construction of the P. ananatis ynfM Gene-Deficient Strain

A PCR reaction was performed using primers as shown in SEQ ID NOs: 205and 206 and pMW118-attL-Tetr-attR (identical to pMW118-attL-Tc-attRplasmid disclosed in WO2005/010175) as a template to amplify a fragmenthaving sequences of 50 bp complementary to the internal sequence of theynfM gene in Pantoea ananatis at the respective ends and containing thetetracycline-resistance gene flanked by the attL and attR sequences of λphage. The SC17(0) strain carrying RSFRedTER was cultured overnight inliquid LB medium, and 1 mL of the culture broth was inoculated into 100mL of liquid LB medium containing final concentrations of 1 mM IPTG and25 mg/L chloramphenicol and incubated with shaking at 34° C. for 3hours. After bacterial cells were collected, they were washed threetimes with 10% glycerol to make competent cells. The amplified PCRfragment was purified using the Wizard PCR Prep manufactured by PromegaCorporation and introduced to the competent cells by electroporation.The electroporation was carried out by using the GENE PULSER II(manufactured by Bio-Rad Laboratories, Inc.) under the followingconditions: electric field intensity, 20 kV/cm; capacitance, 25 μF;electric resistance, 200Ω. The SC17(0) ΔynfM::Tet strain was obtained byselection on LB-agar medium containing 12.5 mg/L of tetracycline.

<3-3> Construction of the P. ananatis sdhA-Deficient Strain

A PCR reaction was performed using primers as shown in SEQ ID NOs: 207and 208 and pMW118-attL-Kmr-attR as a template to amplify a fragmenthaving sequences of 50 bp complementary to the internal sequence of thesdhA gene in Pantoea ananatis at the respective ends and containing thekanamycin-resistance gene flanked by the attL and attR sequences of λphage. The SC17(0) strain carrying RSFRedTER was cultured overnight inliquid LB medium, and 1 mL of the culture broth was inoculated into 100mL of liquid LB medium containing final concentrations of 1 mM IPTG and25 mg/L chloramphenicol and incubated with shaking at 34° C. for 3hours. After bacterial cells were collected, they were washed threetimes with 10% glycerol to make competent cells. The amplified PCRfragment was purified using the Wizard PCR Prep manufactured by PromegaCorporation and introduced to the competent cells by electroporation.The electroporation was carried out by using the GENE PULSER II(manufactured by Bio-Rad Laboratories, Inc.) under the followingconditions: electric field intensity, 20 kV/cm; capacitance, 25 μF;electric resistance, 200Ω. The SC17(0) ΔsdhA::Km strain was obtained byselection on LB-agar medium containing 40 mg/L kanamycin and 20 mMdisodium malate. The obtained strain was applied onto LB-agar mediumcontaining M9 components (17.1 g/L Na₂HPO₄.12H₂O, 3 g/L KH₂PO₄, 0.5 g/LNaCl, 1 g/L NH₄Cl), 10% sucrose, and 1 mM IPTG to obtain a strain whichhad lost the RSFRedTER plasmid. This strain was transfected with thepMW-intxis-sacB plasmid by electroporation and selected on LB-agarmedium containing 25 mg/L chloramphenicol and 20 mM disodium malate toobtain the SC17(0) ΔsdhA::Km/pMW-intxis-sacB strain. After purifyingthis strain on LB-agar medium containing 20 mM disodium malate, areplica was prepared with LB-agar medium containing 40 mg/L kanamycinand 20 mM disodium malate to identify a kanamycin-sensitive strain: theSC17(0) ΔsdhA strain.

<3-4> Construction of Strains Deficient in the yeeA Gene and the ynfMGene from the SC17(0) ΔsdhA Strain

The genomic DNA was extracted from each of the SC17(0) ΔyeeA::Km strainand the SC17(0) ΔynfM::Tet strain by using the Bacterial Genomic DNAPurification Kit manufactured by Edge Biosystems Inc. Separately, theSC17(0) ΔsdhA strain was cultured overnight on LBGM9-agar medium (LBmedium containing 17.1 g/L Na₂HPO₄.12H₂O, 3 g/L KH₂PO₄, 0.5 g/L NaCl, 1g/L NH₄Cl, 5 g/L glucose, 15 g/L agar). Bacterial cells were scrapedusing a cell scraper, washed three times with ice-cold 10% glycerol, andsuspended with 10% glycerol to a final volume of 500 μL to therebyprepare competent cells. To these competent cells, 600 ng of the genomicDNA from the SC17(0) ΔyeeA::Km strain was introduced using the GENEPULSER II (manufactured by Bio-Rad Laboratories, Inc.) under thefollowing conditions: electric field intensity, 20 kV/cm; capacitance,25 μf; electric resistance, 200Ω. After adding ice-cold SOC medium tothe cell suspension and incubating with shaking at 34° C. for 2 hours,transformants were selected at 34° C. on LBGM9-agar medium containing 40mg/L of kanamycin. The obtained strain was named SC17(0) ΔsdhA ΔyeeA::Kmstrain. Similarly, the genomic DNA of the SC17(0) ΔynfM::Tet strain wasintroduced to the host SC17(0) ΔsdhA ΔyeeA::Km strain and the SC17(0)ΔsdhA ΔyeeA::Km ΔynfM::Tet strain was obtained by selection onLBGM9-agar medium containing 12.5 mg/L of tetracycline and 40 mg/L ofkanamycin. These strains were introduced with the pMW-intxis-sacBplasmid by electroporation and selected on LBGM9-agar medium containing25 mg/L of chloramphenicol to obtain the SC17(0) ΔsdhAΔyeeA::Km/pMW-intxis-sacB strain and the SC17(0) ΔsdhA ΔyeeA::KmΔynfM::Tet/pMW-intxis-sacB strain. After purifying the strains onLBGM9-agar medium, replicas were prepared with LBGM9-agar mediumcontaining 40 mg/L of kanamycin or 12.5 mg/L of tetracycline to identifya strain sensitive to kanamycin and a strain sensitive to tetracycline:the SC17(0) ΔsdhA ΔyeeA strain and the SC17(0) ΔsdhA ΔyeeA ΔynfM strain.Competent cells were prepared from the SC17(0) ΔsdhA strain and thesestrains and introduced with the RSFCPG plasmid by electroporation, andtransformants were selected on LBGM9-agar medium containing 12.5 mg/L oftetracycline to obtain the SC17(0) ΔsdhA/RSFCPG strain, the SC17(0)ΔsdhA ΔyeeA/RSFCPG strain, and the SC17(0) ΔsdhA ΔyeeA ΔynfM/RSFCPGstrain.

<3-5> Glutamate Production

Next, the ability to produce glutamic acid was evaluated in thesestrains. The composition of the production medium is shown below.

The composition of the production medium

Part A: Sucrose 100 g/L MgSO₄•7H₂O 0.5 g/L Part B: (NH₄)₂SO₄ 5.0 g/LKH₂PO₄ 6.0 g/L Yeast Extract 6.0 g/L FeSO₄•7H₂O 0.02 g/L MnSO₄•5H₂O 0.02g/L GD113 0.1 mL/L (adjusted to pH 7.0 with KOH)

After Part A and Part B were separately sterilized by autoclaving at120° C. for 20 minutes, they were mixed and tetracycline hydrochloridewas added thereto to 12.5 mg/L.

The SC17(0) ΔsdhA/RSFCPG strain, the SC17(0) ΔsdhA ΔyeeA/RSFCPG strain,and the SC17(0) ΔsdhA ΔyeeA ΔynJM/RSFCPG strain were each culturedovernight at 34° C. on LBGM9-agar medium containing 12.5 mg/L oftetracycline. Bacterial cells on one plate were inoculated into a jarfermenter containing 250 mL of the above-described medium and culturedat 34° C. with stirring at 900 rpm while adjusting pH to 6.0 withammonia.

The results are shown in FIG. 1 and FIG. 2. As compared with the controlSC17(0) ΔsdhA/RSFCPG strain, an increased ability to produce L-glutamicacid was observed in the SC17(0) ΔsdhA ΔyeeA/RSFCPG strain deficient inthe yeeA gene (FIG. 1), and the yield of L-glutamic acid wassignificantly increased in the SC17(0) ΔsdhA ΔyeeA ΔynfM/RSFCPG strainfurther deficient in the ynfM gene (FIG. 2). From the above results, itwas revealed that the ability to produce glutamic acid is increased bydeleting dicarboxylic acid exporter gene(s) also in P. ananatis.

Example 4 Effects of Dicarboxylic Acid Exporter Protein Deficiency inGlutamate Production by Brevibacterium lactofermentum (1)

In this Example, dicarboxylic acid exporter gene-deficient strainsderived from Brevibacterium lactofermentum (Corynebacterium glutamicum)2256 (ATCC 13869) were constructed to produce glutamic acid.

<4-1> Construction of the Brevibacterium Lactofermentum 2256 ΔLdhΔpta-ack ΔpoxB Δach, yggBL30 Strain (FKS0121 Strain)

The L30-type mutation was introduced into the yggB gene on the genome ofthe B. lactofermentum 2256 Δ(ldh, pta-ack, poxB, ach) strain(WO2005/113745) by the following procedures to construct the FKS0121strain having an increased ability to produce glutamic acid.

The pBS4YggB-L plasmid carrying the yggB gene having the L30-typemutation (Japanese Patent Application Publication No. 2007/97573) wasintroduced to the 2256 Δ(ldh, pta-ack, poxB, ach) strain by theelectrical pulse method. The bacterial cells were applied ontoCM-Dex-agar medium containing 25 μg/mL of kanamycin and cultured at31.5° C. A grown strain was verified by PCR to be a single-recombinationstrain in which pBS4yggB-L had been integrated on the genome byhomologous recombination. The single-recombination strain has both thewild-type yggB gene and the mutant yggB gene.

The single-recombination strain was cultured overnight in liquid CM-Dexmedium, and the culture broth was applied onto S10-agar medium (sucrose,100 g/L; polypeptone, 10 g/L; yeast extract, 10 g/L; KH₂PO₄, 1 g/L;MgSO₄.7H₂O, 0.4 g/L; FeSO₄.7H₂O, 0.01 g/L; MnSO₄.4-5H₂O, 0.01 g/L; urea,3 g/L; soy protein hydrolysate solution, 1.2 g/L; agar, 20 g/L; adjustedto pH 7.5 with NaOH; autoclaved at 120° C. for 20 minutes) and incubatedat 31.5° C. Among formed colonies, strains exhibiting kanamycinsensitivity were purified on CM-Dex-agar medium. A PCR was performedusing the genomic DNA prepared from each of these strains and syntheticDNA primers as shown in SEQ ID NO: 251 and SEQ ID NO: 252, and then thenucleotide sequence of the amplified fragment was checked to identify astrain carrying the yggB gene introduced with the L30-type mutation. Thestrain was named FKS0121. The genotype of the strain is 2256 ΔldhΔpta-ack ΔpoxB Δach, yggBL30.

<4-2> Construction of Strains Each Deficient in Succinic Acid ExporterGene Derived from the FKS0121 Strain

<4-2-1> Construction of the FKS0121 ΔsucE1 Strain

The plasmid pBS4S ΔsucE1 for deletion of the sucE1 gene (WO2007/046389)was introduced to the FKS0121 strain by the electrical pulse method. Thebacterial cells were applied onto CM-Dex-agar medium containing 25 μg/mLof kanamycin and cultured at 31.5° C. A grown strain was verified by PCRto be a single-recombination strain in which pBS4S ΔsucE1 had beenintegrated on the genome by homologous recombination. Thesingle-recombination strain has both the wild-type sucE1 gene and thedeletion-type sucE1 gene.

The single-recombination strain was cultured overnight in liquid CM-Dexmedium, and the culture broth was applied onto S10-agar medium (sucrose,100 g/L; polypeptone, 10 g/L; yeast extract, 10 g/L; KH₂PO₄, 1 g/L;MgSO₄.7H₂O, 0.4 g/L; FeSO₄.7H₂O, 0.01 g/L; MnSO₄.4-5H₂O, 0.01 g/L; urea,3 g/L; soy protein hydrolysate solution, 1.2 g/L; agar, 20 g/L; adjustedto pH 7.5 with NaOH; autoclaved at 120° C. for 20 minutes) and incubatedat 31.5° C. Among formed colonies, strains exhibiting kanamycinsensitivity were purified on CM-Dex-agar medium. A PCR was performedusing the genomic DNA prepared from each of these strains and syntheticDNA primers as shown in SEQ ID NO: 253 and SEQ ID NO: 254 to identify astrain deficient in the sucE1 gene. The strain was named FKS0121 ΔsucE1strain.

<4-2-2> Construction of the FKS0121 ΔynfM Strain

Construction of the plasmid pBS4S ΔynfM1 for deletion of the ynfM gene

A PCR was performed using the genomic DNA of the B. lactofermentumstrain 2256 as a template and synthetic DNA primers as shown in SEQ IDNOs: 255 and 256 to obtain a PCR product containing the N-terminalcoding region of the ynfM gene. Separately, a PCR was performed usingthe genomic DNA of the B. lactofermentum strain 2256 as a template andsynthetic DNA primers as shown in SEQ ID NOs: 257 and 258 to obtain aPCR product containing the C-terminal coding region of the ynfM gene.SEQ ID NOs: 256 and 257 partially have complementary sequences. Next,the PCR products containing the N-terminal coding region of the ynfMgene and the C-terminal coding region of the ynfM gene were mixed inalmost equimolar amounts and inserted into the pBS4S vector(WO2007/046389) treated with BamHI and PstI by using the In Fusion HDcloning kit (manufactured by Clontech Laboratories, Inc.). Competentcells of Escherichia coli JM109 (Takara Shuzo Co., Ltd.) weretransformed with this DNA and applied onto LB medium containing 100 μMIPTG 40 μg/mL X-Gal, and 25 μg/mL Km and cultured overnight. Then,formed white colonies were picked up and subjected to single-colonyisolation to obtain transformants. Plasmids were extracted from theobtained transformants to identify a plasmid with the insertion of thePCR product of interest and the plasmid was named pBS4S ΔynfM.

Construction of the FKS0121 ΔynfM Strain

Because the above-obtained pBS4S ΔynfM does not contain a region thatallows the plasmid to replicate autonomously in coryneform bacterialcells, in cases where a coryneform bacterium is transformed with thisplasmid, though very infrequently, a strain carrying this plasmidintegrated on the genome by homologous recombination is formed as atransformant. Thus, the pBS4S ΔynfM1 was introduced to the FKS0121strain by the electrical pulse method. The bacterial cells were appliedonto CM-Dex-agar medium containing 25 μg/mL of kanamycin and cultured at31.5° C. A grown strain was verified by PCR to be a single-recombinationstrain in which pBS4S ΔynfM had been integrated on the genome byhomologous recombination. The single-recombination strain has both thewild-type ynfM gene and the deletion-type ynfM gene.

The single-recombination strain was cultured overnight in liquid CM-Dexmedium, and the culture broth was applied onto S10-agar medium (sucrose,100 g/L; polypeptone, 10 g/L; yeast extract, 10 g/L; KH₂PO₄, 1 g/L;MgSO₄.7H₂O, 0.4 g/L; FeSO₄.7H₂O, 0.01 g/L; MnSO₄.4-5H₂O, 0.01 g/L; urea,3 g/L; soy protein hydrolysate solution, 1.2 g/L; agar, 20 g/L; adjustedto pH 7.5 with NaOH; autoclaved at 120° C. for 20 minutes) and incubatedat 31.5° C. Among formed colonies, strains exhibiting kanamycinsensitivity were purified on CM-Dex-agar medium. A PCR was performedusing the genomic DNA prepared from each of these strains and syntheticDNA primers as shown in SEQ ID NO: 255 and SEQ ID NO: 258 to identify astrain deficient in the ynfM gene. The strain was named FKS0121 ΔynfMstrain.

<4-3> Construction of the FKS0121/pVK9::PmsrA-pyc Strain, the FKS0121ΔsucE1/pVK9::PmsrA-pyc Strain, and the FKS0121 ΔynfM/pVK9::PmsrA-pycStrain

<4-3-1> Construction of the Plasmid pVK9::PmsrA-pyc for Expression ofthe pyc Gene

The plasmid pVK9::PmsrA-pyc for expression of the pyc gene derived fromB. lactofermentum 2256 was produced by the method described below. Thepyc gene is a gene encoding a pyruvate carboxylase. The nucleotidesequence of the pyc gene in B. lactofermentum 2256 and the amino acidsequence of the protein encoded by the same gene are shown in SEQ IDNOs: 275 and 276, respectively. First, the pyc gene was linked bycross-over PCR to the promoter of the methionine sulfoxide reductase Agene (msrA) derived from B. lactofermentum 2256. Specifically, a PCR wasperformed using the genomic DNA of the B. lactofermentum strain 2256 asa template and synthetic DNA primers as shown in SEQ ID NOs: 259 and 260to obtain a PCR product containing the promoter region of the msrA gene.Separately, a PCR was performed using the genomic DNA of the B.lactofermentum strain 2256 as a template and synthetic DNA primers asshown in SEQ ID NOs: 261 and 262 to obtain a PCR product containing theORF region of the pyc gene. SEQ ID NOs: 260 and 261 have complementarysequences. Next, the PCR products containing the promoter region of themsrA gene and the ORF region of the pyc gene were mixed in almostequimolar amounts and a PCR was performed using synthetic DNA primers asshown in SEQ ID NOs: 263 and 264 to obtain the pyc gene fragment linkedto the promoter of the msrA gene. Then, the fragment was inserted intothe pVK9 vector (WO2007/046389) treated with BamHI and PstI by using theIn Fusion HD cloning kit (manufactured by Clontech Laboratories, Inc.).Incidentally, the pVK9 is a shuttle vector for Corynebacterium bacteriaand E. coli. Competent cells of Escherichia coli JM109 (Takara ShuzoCo., Ltd.) were transformed with this DNA and applied onto LB mediumcontaining 100 μM IPTG 40 μg/mL X-Gal, and 25 μg/mL Km and culturedovernight. Then, formed white colonies were picked up and subjected tosingle-colony isolation to obtain transformants. Plasmids were extractedfrom the obtained transformants to identify a plasmid with the insertionof the PCR product of interest and the plasmid was namedpVK9::PmsrA-pyc.

<4-3-2> Introduction of pVK9::PmsrA-pyc

The plasmid pVK9::PmsrA-pyc was introduced to the FKS0121 strain, theFKS0121 ΔsucE1 strain, and the FKS0121 ΔynfM strain by the electricalpulse method. The bacterial cells were applied onto CM-Dex-agar mediumcontaining 25 μg/mL of kanamycin and cultured at 31.5° C. Respectivegrown strains were purified with plates containing the same medium andnamed FKS0121/pVK9::PmsrA-pyc strain, FKS0121 ΔsucE1/pVK9::PmsrA-pycstrain, and FKS0121 ΔynfM/pVK9::PmsrA-pyc strain.

<4-4> Effect of the Deletion of the sucE1 Gene or the ynfM Gene on theProduction of L-Glutamic Acid in the Host FKS0121/pVK9::PmsrA-pyc Strain

The FKS0121/pVK9::PmsrA-pyc strain, the FKS0121 ΔsucE1 I pVK9::PmsrA-pycstrain, and the FKS0121 ΔynfM pVK9::PmsrA-pyc strain, all of which wereobtained by culturing on CM-Dex plating medium, were each inoculatedinto 300 mL of a medium for jar evaluation (glucose, 100 g/L;MgSO₄.7H₂O, 0.5 g/L; H₃PO₄, 2 g/L; soy bean hydrolysate, 1 g/L;(NH₄)₂SO₄, 10 g/L; FeSO₄.7H₂O, 20 mg/L; MnSO₄.5H₂O, 20 mg/L; VB1.HCl, 1mg/L; biotin 3 mg/L; GD-113 (antifoaming agent), 0.65 mL/L; adjusted topH 6.5 with KOH) containing 25 μg/mL of kanamycin and cultured at 31.5°C. for 18 hours using ajar fermenter with 80 mL/min of aeration rate, 20mL/min of CO₂ flow rate, and 300 rpm of stirring speed. Additionally,ammonia was added as appropriate during the culture period so that thepH of the culture was maintained at 6.5.

After the completion of the culture, the concentrations of glutamic acidaccumulated and the sugar remaining in the medium were analyzed with theBiotech Analyzer AS-310 (Sakura SI Co. Ltd.). Moreover, the amount ofsuccinic acid was analyzed with a liquid chromatography HPLC system(L-7100, L-7200, L-7300, or L-7400; Hitachi High-Technologies Co.) andthe URUTRON PS-80H column (Shinwa Chemical Industries Ltd.). Theturbidity (OD) of the bacterial cell suspension was measured using theSpectrophotometer U-2900 (Hitachi).

The results are shown in Table 2. The ability to produce succinic acidwas decreased and the ability to produce L-glutamic acid was increasedin the FKS0121 ΔsucE1/pVK9::PmsrA-pyc strain deficient in the sucE1 geneand the FKS0121 ΔynfM/pVK9::PmsrA-pyc strain deficient in the ynfM geneas compared with the control FKS0121/pVK9::PmsrA-pyc strain. From theabove results, it was revealed that the ability to produce glutamic acidis increased by deleting dicarboxylic acid exporter gene(s) also in thecoryneform bacterium.

TABLE 2 Yield of succinic Yield of glutamic OD620 nm acid (%) acid (%)FKS0121/ 16.5 19.1 9.5 pVK9::PmsrA-pyc FKS0121 ΔsucE1/ 17.1 5.3 10.4pVK9::PmsrA-pyc FKS0121 ΔynfM/ 16.4 17.0 10.4 pVK9::PmsrA-pyc

Example 5 Effects of Dicarboxylic Acid Exporter Protein Deficiency inGlutamate Production by Brevibacterium lactofermentum (2)

In this Example, dicarboxylic acid exporter gene-deficient strainsderived from Brevibacterium lactofermentum (Corynebacterium glutamicum)2256 (ATCC 13869), into which the malyl-CoA pathway was introduced, wereconstructed to produce glutamic acid.

<5-1> Construction of the FKS0121/pVS7::PmsrA-pyc+pVK9::GGMM Strain, theFKS0121 ΔsucE1/pVS7::PmsrA-pyc+pVK9::GGMM Strain, and the FKS0121ΔynfM/pVS7::PmsrA-pyc+pVK9::GGMM Strain

<5-1-1> Construction of the Plasmid pVS7::PmsrA-pyc for Expression ofthe pyc Gene

The plasmid pVS7::PmsrA-pyc for expression of the pyc gene derived fromB. lactofermentum 2256 was produced by the method described below.First, the pyc gene was linked by cross-over PCR to the promoter of themsrA gene derived from B. lactofermentum 2256. Specifically, a PCR wasperformed using the genomic DNA of the B. lactofermentum strain 2256 asa template and synthetic DNA primers as shown in SEQ ID NOs: 259 and 260to obtain a PCR product containing the promoter region of the msrA gene.Separately, a PCR was performed using the genomic DNA of the B.lactofermentum strain 2256 as a template and synthetic DNA primers asshown in SEQ ID NOs: 261 and 262 to obtain a PCR product containing theORF region of the pyc gene. SEQ ID NOs: 260 and 261 have complementarysequences. Next, the PCR products containing the promoter region of themsrA gene and the ORF region of the pyc gene were mixed in almostequimolar amounts and a PCR was performed using synthetic DNA primers asshown in SEQ ID NOs: 263 and 264 to obtain the pyc gene fragment linkedto the promoter of the msrA gene. Then, the fragment was inserted intothe pVS7 vector (WO2013069634) treated with BamHI and PstI by using theIn Fusion HD cloning kit (manufactured by Clontech Laboratories, Inc.).Incidentally, the pVS7 is a shuttle vector for Corynebacterium bacteriaand E. coli. Competent cells of Escherichia coli JM109 (Takara ShuzoCo., Ltd.) were transformed with this DNA and applied onto LB mediumcontaining 100 μM IPTG; 40 μg/mL X-Gal, and 50 μg/mL spectinomycin andcultured overnight. Then, formed white colonies were picked up andsubjected to single-colony isolation to obtain transformants. Plasmidswere extracted from the obtained transformants to identify a plasmidwith the insertion of the PCR product of interest and the plasmid wasnamed pVS7::PmsrA-pyc.

<5-1-2> Construction of the Plasmid pVK9::GGMM for Expression of themalyl-CoA Pathway

The method of introducing the malyl-CoA pathway described in WO2013/018734 is known as a method for increasing Glu flux through thereductive TCA cycle. The malyl-CoA pathway can be introduced byintroducing, for example, the glxR gene, the gcl gene, the mcl gene, andthe mtk gene. The glxR gene is a gene encoding a2-hydroxy-3-oxopropionate reductase, the gcl gene is a gene encoding aglyoxylate carboligase, the mcl gene is a gene encoding a malyl-CoAlyase, and the mtk gene is a gene encoding a malate thiokinase. Thus,the plasmid pVK9::GGMM for expression of the glxR gene, the gcl gene,the mcl gene, and the mtk gene was produced by the method describedbelow. DNA of the glxR-gcl gene region of Rhodococcus jostii (SEQ ID NO:265), and DNA of the lac promoter sequence linked with the mcl-mtk generegion of Methylococcus capsulatus (SEQ ID NO: 266) were each chemicallysynthesized (GenScript Japan Inc.). Next, a PCR was performed using thechemically synthesized DNA as shown in SEQ ID NO: 265 as a template,synthetic DNA primers as shown in SEQ ID NOs: 267 and 268, and PrimeStar polymerase (manufactured by Takara Bio Inc.). Moreover, a PCR wasperformed using the chemically synthesized DNA as shown in SEQ ID NO:266 as a template, synthetic DNA primers as shown in SEQ ID NOs: 269 and270, and Prime Star polymerase (manufactured by Takara Bio Inc.). Ineither case, the reaction solution was adjusted according to thecomposition attached to the kit and the reaction was performed in 30cycles of 98° C. for 10 seconds, 55° C. for 5 seconds, and 72° C. for180 seconds. As a result, a PCR product containing the glxR-gcl genecluster and a PCR product containing the lac promoter sequence linkedwith the mcl-mtk gene cluster were obtained. Furthermore, a PCR wasperformed using pVK9 (WO2007/046389) as a template, synthetic DNAprimers as shown in SEQ ID NOs: 271 and 272, and Prime Star polymerase(manufactured by Takara Bio Inc.). The reaction solution was adjustedaccording to the composition attached to the kit and the reaction wasperformed in 30 cycles of 98° C. for 10 seconds, 55° C. for 5 seconds,and 72° C. for 360 seconds to obtain a PCR product containing thesequence of pVK9. Then, the obtained three PCR products were linkedtogether by using the In-Fusion HD Cloning Kit (manufactured by ClontechLaboratories, Inc.). The obtained plasmid was named pVK9-GGMM.

<5-1-3> Introduction of pVS7::PmsrA-pyc and pVK9::GGMM

The plasmids pVS7::PmsrA-pyc and pVK9::GGMM were introduced to theFKS0121 strain, the FKS0121 ΔsucE1 strain, and the FKS0121 ΔynfM strainby the electrical pulse method. The bacterial cells were applied ontoCM-Dex-agar medium containing 25 μg/mL of kanamycin and 50 μg/mL ofspectinomycin and cultured at 31.5° C. Respective grown strains werepurified with plates containing the same medium and namedFKS0121/pVS7::PmsrA-pyc+pVK9::GGMM strain, FKS0121ΔsucE1/pVS7::PmsrA-pyc+pVK9::GGMM strain, and FKS0121ΔynfM/pVS7::PmsrA-pyc+pVK9::GGMM strain.

<5-2> Effect of the Deletion of the sucE1 Gene or the ynfM Gene on theProduction of L-Glutamic Acid in the HostFKS0121/pVS7::PmsrA-pyc+pVK9::GGMM Strain

The FKS0121/pVS7::PmsrA-pyc+pVK9::GGMM strain, the FKS0121ΔsucE1/pVS7::PmsrA-pyc+pVK9::GGMM strain, and the FKS0121ΔynfM/pVS7::PmsrA-pyc+pVK9::GGMM strain, all of which were obtained byculturing on CM-Dex plating medium, were each inoculated into 3 mL of atest-tube medium (glucose, 20 g/L; urea, 4 g/L; KH₂PO₄, 0.5 g/L; K₂HPO₄0.5 g/L; (NH₄)₂SO₄ 14 g/L; FeSO₄.7H₂O 20 mg/L; MnSO₄.5H₂O 20 mg/L;MgSO₄.7H₂O 0.5 g/L; VB1.HCl 0.2 mg/L; biotin 0.2 mg/L; yeast extract,1/gl; Casamino acids, 1 g/L; no pH adjustment) and cultured with shakingat 31.5° C. for about 16 hours. A 300 μL aliquot of the culture brothwas mixed with 300 μL of a glutamic acid-production medium (glucose, 100g/L; (NH₄)₂SO₄, 2 g/L; HEPES (adjusted to pH 8.2 with KOH), 0.2 M;NaCO₃, 0.2 M) dispensed in a 1.5 mL Eppendorf tube and incubated withshaking under anaerobic conditions at 32° C. for 48 hours.

After the completion of the culture, the concentrations of glutamic acidaccumulated and the sugar remaining in the medium were analyzed with theBiotech Analyzer AS-310 (Sakura SI Co. Ltd.). Moreover, the amount ofsuccinic acid was analyzed with a liquid chromatography HPLC system(L-7100, L-7200, L-7300, or L-7400; Hitachi High-Technologies Co.) andthe URUTRON PS-80H column (Shinwa Chemical Industries Ltd.). Theturbidity (OD) of the bacterial cell suspension was measured using theSpectrophotometer U-2900 (Hitachi).

The results are shown in Table 3. The yield of succinic acid wassignificantly decreased and the yield of glutamic acid was increased byabout 7% in the FKS0121 ΔsucE1/pVS7::PmsrA-pyc+pVK9::GGMM straindeficient in the sucE11 gene as compared with the controlFKS0121/pVS7::PmsrA-pyc+pVK9::GGMM strain. Moreover, any change in theyield of succinic acid was not observed but the yield of L-glutamic acidwas increased by about 2% in the FKS0121ΔynfM/pVS7::PmsrA-pyc+pVK9::GGMM strain deficient in the ynfM gene ascompared with the control FKS0121/pVS7::PmsrA-pyc+pVK9::GGMM strain.From the above results, it was revealed that the ability to produceglutamic acid is increased by deleting dicarboxylic acid exporter genesalso in the bacterial strain into which the malyl-CoA pathway has beenintroduced.

TABLE 3 Yield of succinic Yield of glutamic acid ± S.E. (%) acid ± S.E.(%) FKS0121/ 43 ± 1.7 9.8 ± 0.89 pVS7::PmsrA-pyc + pVK9::GGMM FKS0121ΔsucE1/ 11 ± 4.2  17 ± 1.8  pVS7::PmsrA-pyc + pVK9::GGMM FKS0121 ΔynfM/43 ± 1.5 12 ± 0.77 pVS7::PmsrA-pyc + pVK9::GGMM

Example 6 Effects of Dicarboxylic Acid Exporter Protein Deficiency inGlutamate Production by E. coli (2)

In this Example, dicarboxylic acid exporter gene-deficient strainsderived from E. coli MG1655 (ATCC 47076) were constructed to produceglutamic acid.

<6-1> Construction of the MG1655 ΔsucA ΔgadA ΔgadB ΔiscR Δldh ΔpflDΔpflB ΔptsG Strain

The MG1655 ΔsucA ΔgadA ΔgadB ΔiscR Δldh ΔpflD ΔpflB ΔptsG strain wasconstructed by further deleting the ptsG gene on the genome of theMG1655 ΔsucA ΔgadA ΔgadB ΔiscR Δldh ΔpflD ΔpflB strain obtained inExample <1-2>. This strain was produced according to the methoddescribed in Example <1-2>, i.e. by preparing P1 phage from a ptsGgene-deficient strain in the Keio collection (Baba, T., Ara, T.,Hasegawa, M., Takai, Y, Okumura, Y, Baba, M., Datsenko, K. A., Tomita,M., Wanner, B. L., and Mori, H. (2006) Construction of Escherichia coliK-12 in-frame, single-gene knockout mutants: the Keio collection. MolSyst Biol 2: 2006 0008) and performing P1 transformation to the strainof interest, and subsequent deletion of a drug-resistance gene used as aselection marker.

<6-2> Construction of the MG1655 ΔsucA ΔgadA ΔgadB ΔiscR Δldh ΔpflDΔpflB ΔptsG Δppc Strain

The MG1655 ΔsucA ΔgadA ΔgadB ΔiscR Δldh ΔpflD ΔpflB ΔptsG Δppc strainwas constructed by further deleting the ppc gene on the genome of theMG1655 ΔsucA ΔgadA ΔgadB ΔiscR Δldh ΔpflD ΔpflB ΔptsG strain.

First, MG1655 ΔsucA ΔgadA ΔgadB ΔiscR Δldh ΔpflD ΔpflB Δppc::Cm wasproduced by the λ-Red method. A PCR reaction was performed using primersas shown in SEQ ID NOs: 281 and 282 (each containing a partial sequenceof the ppc gene and either the attL sequence or the attR sequence) andpMW118-attL-Cm-attR as a template. The obtained DNA fragment wasdigested with DpnI restriction enzyme and introduced by electroporationto the MG1655 ΔsucA ΔgadA ΔgadB ΔiscR Δldh ΔpflD ΔpflB strain containingthe plasmid pKD46, which has an ability to replicate in athermo-sensitive manner. Chloramphenicol-resistant recombinants wereselected by culturing at 30° C. on LB-agar medium containing Amp(ampicillin; 50 mg/L) and Cm (chloramphenicol; 20 mg/L). A colony PCRwas performed using primers as shown in SEQ ID NOs: 283 and 284 toselect a strain in which the ppc gene on the genome was replaced withppc::Cm. The selected strain was named MG1655 ΔsucA ΔgadA ΔgadB ΔiscRΔldh ΔpflD ΔpflB Δppc::Cm.

Next, P1 transduction was performed using MG1655 ΔsucA ΔgadA ΔgadB ΔiscRΔldh ΔpflD ΔpflB Δppc::Cm as a donor and MG1655 ΔsucA ΔgadA ΔgadB ΔiscRΔldh ΔpflD ΔpflB ΔptsG as a recipient to obtain MG1655 ΔsucA ΔgadA ΔgadBΔiscR Δldh ΔpflD ΔpflB ΔptsG Δppc::Cm. Then, the pMW-int-xis plasmid wasintroduced to this strain by electroporation, and strains resistant toAmp were selected at 30° C., followed by single-colony isolation of theobtained strains at 42° C. to obtain a strain of interest which had lostthe drug-resistance cassette and the plasmid: the MG1655 ΔsucA ΔgadAΔgadB ΔiscR Δldh ΔpflD ΔpflB ΔptsG Δppc strain.

<6-3> Construction of the MG1655 ΔsucA ΔgadA ΔgadB ΔiscR ΔpflD ΔpflBΔptsG Δppc Δldh::pckA Strain (E7-42 Strain)

A strain carrying the pckA gene linked to the P4071φ10 promoter wasproduced by the λ-Red method, in which strain the pckA gene wasintegrated into the lactate dehydrogenase gene (ldh) locus on the genomeof the MG1655 ΔsucA ΔgadA ΔgadB ΔiscR Δldh ΔpflD ΔpflB ΔptsG Δppcstrain. A PCR reaction was performed using primers as shown in SEQ IDNOs: 285 and 286 (each containing either a partial sequence of theupstream region or downstream region of the ldh gene and either the attLsequence or the attR sequence) and the genome of the Enterobacteraerogenes ES04 ΔpoxB::λattL-Km^(r)-λattR-P4701φ10-pckA strain as atemplate. Incidentally, the ES04 ΔpoxB::λattL-Km^(r)-λattR-P4701φ10-pckAstrain is a strain obtained by replacing the Ptac promoter sequence (SEQID NO: 287) in the ES04 ΔpoxB::λattL-Km^(r)-λattR-Ptac-pckA straindescribed in Example <2-1> with the P4701φ10 promoter sequence (SEQ IDNO: 288). The obtained DNA fragment was digested with DpnI restrictionenzyme and introduced by electroporation to the MG1655 ΔsucA ΔgadA ΔgadBΔiscR ΔpflD ΔpflB ΔptsG Δppc strain containing the plasmid pKD46, whichhas an ability to replicate in a thermo-sensitive manner.Kanamycin-resistant recombinants were selected by culturing at 30° C. onLB-agar medium containing Amp (ampicillin; 50 mg/L) and Km (kanamycin;50 mg/L) to obtain the MG1655 ΔsucA ΔgadA ΔgadB ΔiscR ΔpflD ΔpflB ΔptsGΔppc Δldh::Km-pckA strain. Then, the pMW-int-xis plasmid was introducedto this strain by electroporation, and strains resistant to Amp wereselected at 30° C., followed by single-colony isolation of the obtainedstrains at 42° C. to obtain a strain of interest which had lost thedrug-resistance cassette and the plasmid: the MG1655 ΔsucA ΔgadA ΔgadBΔiscR ΔpflD ΔpflB ΔptsG Δppc Δldh::pckA strain. This strain was namedE7-42 strain.

<6-4> Construction of Strains Each Deficient in Succinic Acid ExporterGene(s) from the E7-42 Strain

<6-4-1> Construction of the E7-42 ΔyjjP Strain

The E7-42 ΔyjjP strain was constructed by further deleting the yjjP geneon the genome of the E7-42 strain. This strain was produced according tothe method described in Example <1-2>, i.e. by preparing P1 phage from ayjjP gene-deficient strain in the Keio collection (Baba, T., Ara, T.,Hasegawa, M., Takai, Y, Okumura, Y, Baba, M., Datsenko, K. A., Tomita,M., Wanner, B. L., and Mori, H. (2006) Construction of Escherichia coliK-12 in-frame, single-gene knockout mutants: the Keio collection. MolSyst Biol 2: 2006 0008) and performing P1 transformation to the E7-42strain, and subsequent deletion of a drug-resistance gene used as aselection marker.

<6-4-2> Construction of the E7-42 ΔyjjP ΔyeeA ΔynfM Strain

The E7-42 ΔyjjP ΔyeeA ΔynfM strain was constructed by further deletingthe yeeA and ynfM genes on the genome of the E7-42 ΔyjjP strain.

First, P1 transduction was performed using MG1655 ΔyeeA::Cm (describedin Example <1-5>) as a donor and the E7-42 ΔyjjP strain as a recipientto obtain E7-42 ΔyjjP ΔyeeA::Cm. Then, the pMW-int-xis plasmid wasintroduced to this strain by electroporation, and strains resistant toAmp were selected at 30° C., followed by single-colony isolation of theobtained strains at 42° C. to obtain a strain of interest which had lostthe drug-resistance cassette and the plasmid: the E7-42 ΔyjjP ΔyeeAstrain.

Next, P1 transduction was performed using MG1655 ΔynJM::Cm (described inExample <1-5>) as a donor and the E7-42 ΔyjjP ΔyeeA strain as arecipient to obtain E7-42 ΔyjjP ΔyeeA ΔynfM::Cm. Then, the pMW-int-xisplasmid was introduced to this strain by electroporation, and strainsresistant to Amp were selected at 30° C., followed by single-colonyisolation of the obtained strains at 42° C. to obtain a strain ofinterest which had lost the drug-resistance cassette and the plasmid:the E7-42 ΔyjjP ΔyeeA ΔynfM strain.

<6-5> Confirmation of Glutamate Production in Anaerobic Culture

Strains were produced by simultaneously introducing the RSFCPG plasmid(see EP0952221) and the pMW219-FdKGSPS plasmid (described in Example<1-6>) to each of the strains E7-42, E7-42 ΔyjjP and E7-42 ΔyjjP ΔyeeAΔynfM

Next, the L-glutamate production culture was performed by using theproduced bacterial strains to examine the ability to produce L-glutamicacid. The bacterial strains were uniformly applied onto LBM9Glc plates(produced by adding 200 mL of 5×M9 salts and 10 mL of 50% glucose to 800mL of LB medium) containing a suitable antibiotic (50 μg/mL of kanamycinor 15 μg/mL of tetracycline) and cultured at 37° C. for 16-20 hours.Then, those plates were placed into an AnaeroPack pouch (manufactured byMitsubishi Gas Chemical Company, Inc.; for easy cultivation of anaerobicbacteria; Product Number: A-04) and incubated under anaerobic conditionsat 37° C. for 6 hours. Obtained bacterial cells on the plates weresuspended in 700 μL of 0.8% saline to give an optical density (OD) of0.5 to 1.5 (600 nm) when diluted 51 times. To a microtube having avolume of 1.5 mL, 200 μl of this bacterial cell suspension and 1 mL ofthe production medium purged with a sufficient amount of carbon dioxidegas in advance (1 vvm, 30 minutes or longer) were placed and coveredtightly with a cap and then incubated using a microtube shaker underanaerobic conditions at 37° C. for 24 or 48 hours. The composition ofthe production medium is shown below.

The composition of the production medium

Part A: Glucose 10 g/L (final concentration) Part B: Magnesium sulfateheptahydrate 1 g/L Ammonium sulfate 15 g/L Monopotassium phosphate 1 g/LBiotin 1 mg/L Vitamin B1 1 mg/L FeSO₄•7H₂O 10 mg/L (adjusted to pH = 7with KOH) Part C: Calcium carbonate (Japanese Pharmacopeia) 50 g/L

After Part A and Part B were separately sterilized by autoclaving at115° C. for 10 minutes and Part C was sterilized by dry-heating at 180°C. for 3 hours, they were left to cool and then mixed.

After the culture, the concentrations of glutamic acid accumulated andthe sugar remaining in the medium were analyzed with the BiotechAnalyzer AS-310 (Sakura SI Co. Ltd.). Moreover, the amounts of otherorganic acids were analyzed with a liquid chromatography HPLC system(L-7100, L-7200, L-7300, or L-7400; Hitachi High-Technologies Co.) andthe URUTRON PS-80H column (Shinwa Chemical Industries Ltd.). Theturbidity (OD) of the bacterial cell suspension was measured using theSpectrophotometer DU800 (Beckman Coulter) after calcium carbonate in themedium was dissolved by diluting the sample with 0.1 N hydrochloricacid.

The results are shown in Table 4. The ability to produce L-glutamic acidwas increased in the E7-42 ΔyjjP (RSFCPG, pMW219-FdKGSPS) straindeficient in the yjjP gene and in the E7-42 ΔyjjP ΔyeeA ΔynfM (RSFCPG,pMW219-FdKGSPS) strain further deficient in the ynfM and yeeA genes ascompared with the control E7-42 (RSFCPG, pMW219-FdKGSPS) strain. Fromthe above results, it was revealed that the ability to produce glutamicacid is increased by deleting dicarboxylic acid exporter gene(s) also inE. coli.

TABLE 4 Yield of glutamic acid ± S.E. Yield of succinic acid ± Strainsto be evaluated (*1) (%) S.E. (%) E7-42 5.7 ± 0.2 54.3 ± 1.5 E7-42 ΔyjjP6.3 ± 0.2 42.4 ± 2.5 E7-42 ΔyjjP ΔynfM ΔyeeA 7.9 ± 0.3 40.1 ± 1.4 (*1)each strain carries both RSFCPG and pMW219-FdKGSPS plasmids.

INDUSTRIAL APPLICABILITY

According to the present invention, the ability of a microorganism toproduce an objective substance can be improved, and the objectivesubstance can be produced efficiently.

<Explanation of Sequence Listing>

SEQ ID NO: 1: Nucleotide sequence of nuo operon of E. coli MG1655

SEQ ID NOS: 2 to 14: Amino acid sequences of proteins encoded by nuooperon of E. coli MG1655

SEQ ID NO: 15: Nucleotide sequence of nuo operon of Pantoea ananatisAJ13355

SEQ ID NOS: 16 to 28: Amino acid sequences of proteins encoded by nuooperon of Pantoea ananatis AJ13355

SEQ ID NO: 29: Nucleotide sequence of ndh gene of E. coli MG1655

SEQ ID NO: 30: Amino acid sequence of Ndh protein of E. coli MG1655

SEQ ID NO: 31: Nucleotide sequence of ndh gene of Pantoea ananatisAJ13355

SEQ ID NO: 32: Amino acid sequence of Ndh protein of Pantoea ananatisAJ13355

SEQ ID NO: 33: Nucleotide sequence of mqo gene of E. coli MG1655

SEQ ID NO: 34: Amino acid sequence of Mqo protein of E. coli MG1655

SEQ ID NO: 35: Nucleotide sequence of mqo1 gene of Pantoea ananatisAJ13355

SEQ ID NO: 36: Amino acid sequence of a protein encoded by mqo1 gene ofPantoea ananatis AJ13355

SEQ ID NO: 37: Nucleotide sequence of ldhA gene of E. coli MG1655

SEQ ID NO: 38: Amino acid sequence of LdhA protein of E. coli MG1655

SEQ ID NO: 39: Nucleotide sequence of ldhA gene of Pantoea ananatisAJ13355

SEQ ID NO: 40: Amino acid sequence of LdhA protein of Pantoea ananatisAJ13355

SEQ ID NO: 41: Nucleotide sequence of adhE gene of E. coli MG1655

SEQ ID NO: 42: Amino acid sequence of AdhE protein of E. coli MG1655

SEQ ID NO: 43: Nucleotide sequence of adhE gene of Pantoea ananatisAJ13355

SEQ ID NO: 44: Amino acid sequence of AdhE protein of Pantoea ananatisAJ13355

SEQ ID NO: 45: Nucleotide sequence of pta gene of E. coli MG1655

SEQ ID NO: 46: Amino acid sequence of Pta protein of E. coli MG1655

SEQ ID NO: 47: Nucleotide sequence of pta gene of Pantoea ananatisAJ13355

SEQ ID NO: 48: Amino acid sequence of Pta protein of Pantoea ananatisAJ13355

SEQ ID NO: 49: Nucleotide sequence of α-subunit gene of α-ketoglutaratesynthase of Chlorobium tepidum

SEQ ID NO: 50: Amino acid sequence of α-subunit of α-ketoglutaratesynthase of Chlorobium tepidum

SEQ ID NO: 51: Nucleotide sequence of β-subunit gene of α-ketoglutaratesynthase of Chlorobium tepidum

SEQ ID NO: 52: Amino acid sequence of β-subunit of α-ketoglutaratesynthase of Chlorobium tepidum

SEQ ID NO: 53: Nucleotide sequence of α-subunit gene of α-ketoglutaratesynthase of Blastopirellula marina

SEQ ID NO: 54: Amino acid sequence of α-subunit of α-ketoglutaratesynthase of Blastopirellula marina

SEQ ID NO: 55: Nucleotide sequence of β-subunit gene of α-ketoglutaratesynthase of Blastopirellula marina

SEQ ID NO: 56: Amino acid sequence of β-subunit of α-ketoglutaratesynthase of Blastopirellula marina

SEQ ID NO: 57: Nucleotide sequence of fpr gene of E. coli K-12

SEQ ID NO: 58: Amino acid sequence of Fpr protein of E. coli K-12

SEQ ID NO: 59: Nucleotide sequence of pyruvate synthase gene ofChlorobium tepidum

SEQ ID NO: 60: Amino acid sequence of pyruvate synthase of Chlorobiumtepidum

SEQ ID NO: 61: Nucleotide sequence of fdx gene of E. coli K-12

SEQ ID NO: 62: Amino acid sequence of Fdx protein of E. coli K-12

SEQ ID NO: 63: Nucleotide sequence of yfhL gene of E. coli K-12

SEQ ID NO: 64: Amino acid sequence of YfhL protein of E. coli K-12

SEQ ID NO: 65: Nucleotide sequence of fldA gene of E. coli K-12

SEQ ID NO: 66: Amino acid sequence of FldA protein of E. coli K-12

SEQ ID NO: 67: Nucleotide sequence of fldB gene of E. coli K-12

SEQ ID NO: 68: Amino acid sequence of FldB protein of E. coli K-12

SEQ ID NO: 69: Nucleotide sequence of ferredoxin I gene of Chlorobiumtepidum

SEQ ID NO: 70: Amino acid sequence of ferredoxin I of Chlorobium tepidum

SEQ ID NO: 71: Nucleotide sequence of ferredoxin II gene of Chlorobiumtepidum

SEQ ID NO: 72: Amino acid sequence of ferredoxin II of Chlorobiumtepidum

SEQ ID NO: 73: Nucleotide sequence of sucA gene of Pantoea ananatisAJ13355

SEQ ID NO: 74: Amino acid sequence of SucA protein of Pantoea ananatisAJ13355

SEQ ID NO: 75: Nucleotide sequence of sucB gene of Pantoea ananatisAJ13355

SEQ ID NO: 76: Amino acid sequence of SucB protein of Pantoea ananatisAJ13355

SEQ ID NO: 77: Nucleotide sequence of lpdA gene of Pantoea ananatisAJ13355

SEQ ID NO: 78: Amino acid sequence of LpdA protein of Pantoea ananatisAJ13355

SEQ ID NO: 79: Nucleotide sequence of odhA gene of Corynebacteriumglutamicum ATCC13032

SEQ ID NO: 80: Amino acid sequence of E1o subunit of Corynebacteriumglutamicum ATCC13032

SEQ ID NO: 81: Nucleotide sequence of lpd gene of Corynebacteriumglutamicum ATCC13032

SEQ ID NO: 82: Amino acid sequence of E3 subunit of Corynebacteriumglutamicum ATCC13032

SEQ ID NO: 83: Nucleotide sequence of NCgl2126 gene of Corynebacteriumglutamicum ATCC13032

SEQ ID NO: 84: Amino acid sequence of a protein encoded by NCgl2126 geneof Corynebacterium glutamicum ATCC13032

SEQ ID NO: 85: Nucleotide sequence of ndh gene of Corynebacteriumglutamicum ATCC13032

SEQ ID NO: 86: Amino acid sequence of Ndh protein of Corynebacteriumglutamicum ATCC13032

SEQ ID NO: 87: Nucleotide sequence of mqo gene of Corynebacteriumglutamicum ATCC13032

SEQ ID NO: 88: Amino acid sequence of Mqo protein of Corynebacteriumglutamicum ATCC13032

SEQ ID NO: 89: Nucleotide sequence of pflB gene of E. coli MG1655

SEQ ID NO: 90: Amino acid sequence of PflB protein of E. coli MG1655

SEQ ID NO: 91: Nucleotide sequence of pflD gene of E. coli MG1655

SEQ ID NO: 92: Amino acid sequence of PflD protein of E. coli MG1655

SEQ ID NO: 93: Nucleotide sequence of tdcE gene of E. coli MG1655

SEQ ID NO: 94: Amino acid sequence of TdcE protein of E. coli

MG1655

SEQ ID NO: 95: Nucleotide sequence of pflB gene of Pantoea ananatisAJ13355

SEQ ID NO: 96: Amino acid sequence of PflB protein of Pantoea ananatisAJ13355

SEQ ID NO: 97: Nucleotide sequence of mqo2 gene of Pantoea ananatisAJ13355

SEQ ID NO: 98: Amino acid sequence of a protein encoded by mqo2 gene ofPantoea ananatis AJ13355

SEQ ID NO: 99: Nucleotide sequence of mtkA gene of Methylobacteriumextorquens AM1

SEQ ID NO: 100: Amino acid sequence of MtkA protein of Methylobacteriumextorquens AM1

SEQ ID NO: 101: Nucleotide sequence of mtkB gene of Methylobacteriumextorquens AM1

SEQ ID NO: 102: Amino acid sequence of MtkB protein of Methylobacteriumextorquens AM1

SEQ ID NO: 103: Nucleotide sequence of mtkA gene of Mesorhizobium lotiMAFF303099

SEQ ID NO: 104: Amino acid sequence of MtkA protein of Mesorhizobiumloti MAFF303099

SEQ ID NO: 105: Nucleotide sequence of mtkB gene of Mesorhizobium lotiMAFF303099

SEQ ID NO: 106: Amino acid sequence of MtkB protein of Mesorhizobiumloti MAFF303099

SEQ ID NO: 107: Nucleotide sequence of mtkA gene of Granulibacterbethesdensis CGDNIH1

SEQ ID NO: 108: Amino acid sequence of MtkA protein of Granulibacterbethesdensis CGDNIH1

SEQ ID NO: 109: Nucleotide sequence of mtkB gene of Granulibacterbethesdensis CGDNIH1

SEQ ID NO: 110: Amino acid sequence of MtkB protein of Granulibacterbethesdensis CGDNIH1

SEQ ID NO: 111: Nucleotide sequence of sucC gene of E. coli MG1655

SEQ ID NO: 112: Amino acid sequence of SucC protein of E. coli MG1655

SEQ ID NO: 113: Nucleotide sequence of sucD gene of E. coli MG1655

SEQ ID NO: 114: Amino acid sequence of SucD protein of E. coli MG1655

SEQ ID NO: 115: Nucleotide sequence of sucC gene of Pantoea ananatisAJ13355

SEQ ID NO: 116: Amino acid sequence of SucC protein of Pantoea ananatisAJ13355

SEQ ID NO: 117: Nucleotide sequence of sucD gene of Pantoea ananatisAJ13355

SEQ ID NO: 118: Amino acid sequence of SucD protein of Pantoea ananatisAJ13355

SEQ ID NO: 119: Nucleotide sequence of sucC gene of Corynebacteriumglutamicum ATCC13032

SEQ ID NO: 120: Amino acid sequence of SucC protein of Corynebacteriumglutamicum ATCC13032

SEQ ID NO: 121: Nucleotide sequence of sucD gene of Corynebacteriumglutamicum ATCC13032

SEQ ID NO: 122: Amino acid sequence of SucD protein of Corynebacteriumglutamicum ATCC13032

SEQ ID NO: 123: Nucleotide sequence of sucC gene of Corynebacteriumglutamicum 2256 (ATCC13869)

SEQ ID NO: 124: Amino acid sequence of SucC protein of Corynebacteriumglutamicum 2256 (ATCC13869)

SEQ ID NO: 125: Nucleotide sequence of sucD gene of Corynebacteriumglutamicum 2256 (ATCC13869)

SEQ ID NO: 126: Amino acid sequence of SucD protein of Corynebacteriumglutamicum 2256 (ATCC13869)

SEQ ID NO: 127: Nucleotide sequence of Ca_smtA gene of Chloroflexusaurantiacus J-10-fl

SEQ ID NO: 128: Amino acid sequence of Ca_SmtA protein of Chloroflexusaurantiacus J-10-fl

SEQ ID NO: 129: Nucleotide sequence of Ca_smtB gene of Chloroflexusaurantiacus J-10-fl

SEQ ID NO: 130: Amino acid sequence of Ca_SmtB protein of Chloroflexusaurantiacus J-10-fl

SEQ ID NO: 131: Nucleotide sequence of Ap_smtA gene of CandidatusAccumulibacter phosphatis clade IIA str. UW-1

SEQ ID NO: 132: Amino acid sequence of Ap_SmtA protein of CandidatusAccumulibacter phosphatis clade IIA str. UW-1

SEQ ID NO: 133: Nucleotide sequence of Ap_smtB gene of CandidatusAccumulibacter phosphatis clade IIA str. UW-1

SEQ ID NO: 134: Amino acid sequence of Ap_SmtB protein of CandidatusAccumulibacter phosphatis clade IIA str. UW-1

SEQ ID NO: 135: Nucleotide sequence of Rr_smt gene of Rhodospirillumrubrum ATCC 11170

SEQ ID NO: 136: Amino acid sequence of Rr_Smt protein of Rhodospirillumrubrum ATCC 11170

SEQ ID NO: 137: Nucleotide sequence of Mm_smt gene of Magnetospirillummagneticum AMB-1

SEQ ID NO: 138: Amino acid sequence of Mm_Smt protein ofMagnetospirillum magneticum AMB-1

SEQ ID NO: 139: Nucleotide sequence of mclA gene of Methylobacteriumextorquens AM1

SEQ ID NO: 140: Amino acid sequence of MclA protein of Methylobacteriumextorquens AM1

SEQ ID NO: 141: Nucleotide sequence of mclA gene of Mesorhizobium lotiMAFF303099

SEQ ID NO: 142: Amino acid sequence of MclA protein of Mesorhizobiumloti MAFF303099

SEQ ID NO: 143: Nucleotide sequence of mclA gene of Granulibacterbethesdensis CGDNIH1

SEQ ID NO: 144: Amino acid sequence of MclA protein of Granulibacterbethesdensis CGDNIH1

SEQ ID NO: 145: Nucleotide sequence of aceA gene of E. coli MG1655

SEQ ID NO: 146: Amino acid sequence of AceA protein of E. coli MG1655

SEQ ID NO: 147: Nucleotide sequence of aceA gene of Pantoea ananatisAJ13355

SEQ ID NO: 148: Amino acid sequence of AceA protein of Pantoea ananatisAJ13355

SEQ ID NO: 149: Nucleotide sequence of ICL1 gene of Corynebacteriumglutamicum ATCC13032

SEQ ID NO: 150: Amino acid sequence of a protein encoded by ICL1 gene ofCorynebacterium glutamicum ATCC13032

SEQ ID NO: 151: Nucleotide sequence of ICL2 gene of Corynebacteriumglutamicum ATCC13032

SEQ ID NO: 152: Amino acid sequence of a protein encoded by ICL2 gene ofCorynebacterium glutamicum ATCC13032

SEQ ID NO: 153: Nucleotide sequence of ICL1 gene of Corynebacteriumglutamicum 2256 (ATCC13869)

SEQ ID NO: 154: Amino acid sequence of a protein encoded by ICL1 gene ofCorynebacterium glutamicum 2256 (ATCC13869)

SEQ ID NO: 155: Nucleotide sequence of ICL2 gene of Corynebacteriumglutamicum 2256 (ATCC13869)

SEQ ID NO: 156: Amino acid sequence of a protein encoded by ICL2 gene ofCorynebacterium glutamicum 2256 (ATCC13869)

SEQ ID NO: 157: Nucleotide sequence of yjjP gene of E. coli MG1655

SEQ ID NO: 158: Amino acid sequence of YjjP protein of E. coli MG1655

SEQ ID NO: 159: Nucleotide sequence of yjjP gene of Enterobacteraerogenes

SEQ ID NO: 160: Amino acid sequence of YjjP protein of Enterobacteraerogenes

SEQ ID NO: 161: Nucleotide sequence of yjjB gene of E. coli MG1655

SEQ ID NO: 162: Amino acid sequence of YjjB protein of E. coli MG1655

SEQ ID NO: 163: Nucleotide sequence of yjjB gene of Enterobacteraerogenes

SEQ ID NO: 164: Amino acid sequence of YjjB protein of Enterobacteraerogenes

SEQ ID NO: 165: Nucleotide sequence of yeeA gene of E. coli MG1655

SEQ ID NO: 166: Amino acid sequence of YeeA protein of E. coli MG1655

SEQ ID NO: 167: Nucleotide sequence of yeeA gene of Pantoea ananatisAJ13355

SEQ ID NO: 168: Amino acid sequence of YeeA protein of Pantoea ananatisAJ13355

SEQ ID NO: 169: Nucleotide sequence of yeeA gene of Enterobacteraerogenes

SEQ ID NO: 170: Amino acid sequence of YeeA protein of Enterobacteraerogenes

SEQ ID NO: 171: Nucleotide sequence of ynfM gene of E. coli MG1655

SEQ ID NO: 172: Amino acid sequence of YnfM protein of E. coli MG1655

SEQ ID NO: 173: Nucleotide sequence of ynfM gene of Pantoea ananatisAJ13355

SEQ ID NO: 174: Amino acid sequence of YnfM protein of Pantoea ananatisAJ13355

SEQ ID NO: 175: Nucleotide sequence of ynfM gene of Enterobacteraerogenes

SEQ ID NO: 176: Amino acid sequence of YnfM protein of Enterobacteraerogenes

SEQ ID NO: 177: Nucleotide sequence of ynfM gene of Corynebacteriumglutamicum ATCC13032

SEQ ID NO: 178: Amino acid sequence of YnfM protein of Corynebacteriumglutamicum ATCC13032

SEQ ID NO: 179: Nucleotide sequence of ynfM gene of Corynebacteriumglutamicum 2256 (ATCC13869)

SEQ ID NO: 180: Amino acid sequence of YnfM protein of Corynebacteriumglutamicum 2256 (ATCC13869)

SEQ ID NOS: 181 to 212: Primers

SEQ ID NO: 213: pckA gene of Actinobacillus succinogenes 130Z

SEQ ID NO: 214: PckA protein of Actinobacillus succinogenes 130Z

SEQ ID NOS: 215 to 228: Primers

SEQ ID NO: 229: Nucleotide sequence of ldh gene of Corynebacteriumglutamicum ATCC13032

SEQ ID NO: 230: Amino acid sequence of Ldh protein of Corynebacteriumglutamicum ATCC13032

SEQ ID NO: 231: Nucleotide sequence of ldh gene of Corynebacteriumglutamicum 2256 (ATCC13869)

SEQ ID NO: 232: Amino acid sequence of Ldh protein of Corynebacteriumglutamicum 2256 (ATCC13869)

SEQ ID NO: 233: Nucleotide sequence of adhE gene of Corynebacteriumglutamicum ATCC13032

SEQ ID NO: 234: Amino acid sequence of AdhE protein of Corynebacteriumglutamicum ATCC13032

SEQ ID NO: 235: Nucleotide sequence of ilvB gene of E. coli MG1655

SEQ ID NO: 236: Amino acid sequence of IlvB protein of E. coli MG1655

SEQ ID NO: 237: Nucleotide sequence of ilvI gene of E. coli MG1655

SEQ ID NO: 238: Amino acid sequence of IlvI protein of E. coli MG1655

SEQ ID NO: 239: Nucleotide sequence of ilvG gene of Pantoea ananatisAJ13355

SEQ ID NO: 240: Amino acid sequence of IlvG protein of Pantoea ananatisAJ13355

SEQ ID NO: 241: Nucleotide sequence of ilvI gene of Pantoea ananatisAJ13355

SEQ ID NO: 242: Amino acid sequence of IlvI protein of Pantoea ananatisAJ13355

SEQ ID NO: 243: Nucleotide sequence of ilvB gene of Corynebacteriumglutamicum ATCC13032

SEQ ID NO: 244: Amino acid sequence of IlvB protein of Corynebacteriumglutamicum ATCC13032

SEQ ID NO: 245: Nucleotide sequence of budA gene of Pantoea ananatisAJ13355

SEQ ID NO: 246: Amino acid sequence of BudA protein of Pantoea ananatisAJ13355

SEQ ID NO: 247: Nucleotide sequence of budC gene of Pantoea ananatisAJ13355

SEQ ID NO: 248: Amino acid sequence of BudC protein of Pantoea ananatisAJ13355

SEQ ID NO: 249: Nucleotide sequence of butA gene of Corynebacteriumglutamicum ATCC13032

SEQ ID NO: 250: Amino acid sequence of ButA protein of Corynebacteriumglutamicum ATCC13032

SEQ ID NOS: 251 to 264: Primers

SEQ ID NO: 265: Nucleotide sequence of glxR-glc gene region ofRhodococcus jostii

SEQ ID NO: 266: Nucleotide sequence of lac promoter region and mcl-mtkgene region of Methylococcus capsulatus

SEQ ID NOS: 267 to 272: Primers

SEQ ID NO: 273: Nucleotide sequence of yggB gene of Corynebacteriumglutamicum 2256 (ATCC13869)

SEQ ID NO: 274: Amino acid sequence of YggB protein of Corynebacteriumglutamicum 2256 (ATCC13869)

SEQ ID NO: 275: Nucleotide sequence of pyc gene of Corynebacteriumglutamicum 2256 (ATCC13869)

SEQ ID NO: 276: Amino acid sequence of Pyc protein of Corynebacteriumglutamicum 2256 (ATCC13869)

SEQ ID NO: 277: Nucleotide sequence of sucE1 gene of Corynebacteriumglutamicum ATCC13032

SEQ ID NO: 278: Amino acid sequence of SucE1 protein of Corynebacteriumglutamicum ATCC13032

SEQ ID NO: 279: Nucleotide sequence of sucE1 gene of Corynebacteriumglutamicum 2256 (ATCC13869)

SEQ ID NO: 280: Amino acid sequence of SucE1 protein of Corynebacteriumglutamicum 2256 (ATCC13869)

SEQ ID NOS: 281 to 286: Primers

SEQ ID NO: 287: Ptac promoter

SEQ ID NO: 288: P4701φ10 promoter

1. A method for producing an objective substance, the method comprising: culturing a microorganism having an objective substance-producing ability in a medium to produce and accumulate the objective substance in the medium or in cells of the microorganism; and collecting the objective substance from the medium or the cells, wherein the microorganism has been modified so that the activity of a dicarboxylic acid exporter protein is reduced.
 2. The method according to claim 1, wherein the activity of the dicarboxylic acid exporter protein is reduced by attenuating the expression of a gene encoding the dicarboxylic acid exporter protein or by deleting the gene.
 3. The method according to claim 2, wherein the gene encoding the dicarboxylic acid exporter protein is selected from the group consisting of yjjP gene, yjjB gene, yeeA gene, ynfM gene, sucE1 gene, and combinations thereof.
 4. The method according to claim 3, wherein the yjjP gene is a DNA selected from the group consisting of: (A) a DNA encoding a protein comprising the amino acid sequence of SEQ ID NO: 158 or 160; (B) a DNA encoding a protein comprising the amino acid sequence of SEQ ID NO: 158 or 160, but including substitution, deletion, insertion, or addition of one or several amino acid residues, the protein having an activity to export a dicarboxylic acid; (C) a DNA comprising the nucleotide sequence of SEQ ID NO: 157 or 159; and (D) a DNA able to hybridize under stringent conditions with a nucleotide sequence complementary to the nucleotide sequence of SEQ ID NO: 157 or 159, or with a probe that can be prepared from the complementary nucleotide sequence, and encoding a protein having an activity to export a dicarboxylic acid.
 5. The method according to claim 3, wherein the yjjB gene is a DNA selected from the group consisting of: (A) DNA encoding a protein comprising the amino acid sequence of SEQ ID NO: 162 or 164; (B) DNA encoding a protein comprising the amino acid sequence of SEQ ID NO: 162 or 164, but including substitution, deletion, insertion, or addition of one or several amino acid residues, the protein having an activity to export a dicarboxylic acid; (C) DNA comprising the nucleotide sequence of SEQ ID NO: 161 or 163; and (D) DNA able to hybridize under stringent conditions with a nucleotide sequence complementary to the nucleotide sequence of SEQ ID NO: 161 or 163, or with a probe that can be prepared from the complementary nucleotide sequence, and encoding a protein having an activity to export a dicarboxylic acid.
 6. The method according to claim 3, wherein the yeeA gene is a DNA selected from the group consisting of: (A) DNA encoding a protein comprising the amino acid sequence of SEQ ID NO: 166, 168, or 170; (B) DNA encoding a protein comprising the amino acid sequence of SEQ ID NO: 166, 168, or 170, but including substitution, deletion, insertion, or addition of one or several amino acid residues, the protein having an activity to export a dicarboxylic acid; (C) DNA comprising the nucleotide sequence of SEQ ID NO: 165, 167, or 169; and (D) DNA able to hybridize under stringent conditions with a nucleotide sequence complementary to the nucleotide sequence of SEQ ID NO: 165, 167, or 169, or with a probe that can be prepared from the complementary nucleotide sequence, and encoding a protein having an activity to export a dicarboxylic acid.
 7. The method according to claim 3, wherein the ynfM gene is a DNA selected from the group consisting of: (A) DNA encoding a protein comprising the amino acid sequence of SEQ ID NO: 172, 174, 176, 178, or 180; (B) DNA encoding a protein comprising the amino acid sequence of SEQ ID NO: 172, 174, 176, 178, or 180, but including substitution, deletion, insertion, or addition of one or several amino acid residues, the protein having an activity to export a dicarboxylic acid; (C) DNA comprising the nucleotide sequence of SEQ ID NO: 171, 173, 175, 177, or 179; and (D) DNA able to hybridize under stringent conditions with a nucleotide sequence complementary to the nucleotide sequence of SEQ ID NO: 171, 173, 175, 177, or 179, or with a probe that can be prepared from the complementary nucleotide sequence, and encoding a protein having an activity to export a dicarboxylic acid.
 8. The method according to claim 3, wherein the sucE1 gene is a DNA selected from the group consisting of: (A) DNA encoding a protein comprising the amino acid sequence of SEQ ID NO: 278 or 280; (B) DNA encoding a protein comprising the amino acid sequence of SEQ ID NO: 278 or 280, but including substitution, deletion, insertion, or addition of one or several amino acid residues, the protein having an activity to export a dicarboxylic acid; (C) DNA comprising the nucleotide sequence of SEQ ID NO: 277 or 279; and (D) DNA able to hybridize under stringent conditions with a nucleotide sequence complementary to the nucleotide sequence of SEQ ID NO: 277 or 279, or with a probe that can be prepared from the complementary nucleotide sequence, and encoding a protein having an activity to export a dicarboxylic acid.
 9. The method according to claim 1, wherein the objective substance is a metabolite derived from acetyl-CoA and/or an L-amino acid.
 10. The method according to claim 9, wherein the metabolite derived from acetyl-CoA and/or the L-amino acid is selected from the group consisting of isopropyl alcohol, ethanol, acetone, propylene, isoprene, 1,3-butanediol, 1,4-butanediol, 1-propanol, 1,3-propanediol, 1,2-propanediol, ethylene glycol, isobutanol, and combinations thereof.
 11. The method according to claim 9, wherein the metabolite derived from acetyl-CoA and/or the L-amino acid is selected from the group consisting of citric acid, itaconic acid, acetic acid, butyric acid, 3-hydroxybutyric acid, polyhydroxybutyric acid, 3-hydroxyisobutyric acid, 3-aminoisobutyric acid, 2-hydroxyisobutyric acid, methacrylic acid, 6-aminocaproic acid, and combinations thereof.
 12. The method according to claim 9, wherein the metabolite derived from acetyl-CoA and/or the L-amino acid is selected from the group consisting of polyglutamic acid, L-glutamic acid, L-glutamine, L-arginine, L-ornithine, L-citrulline, L-leucine, L-isoleucine, L-valine, L-cysteine, L-serine, L-proline, and combinations thereof.
 13. The method according to claim 12, wherein the L-glutamic acid is monoammonium L-glutamate or monosodium L-glutamate.
 14. The method according to claim 1, wherein the microorganism is a coryneform bacterium or a bacterium belonging to the family Enterobacteriaceae.
 15. The method according to claim 14, wherein the coryneform bacterium is Corynebacterium glutamicum.
 16. The method according to claim 14, wherein the bacterium belonging to the family Enterobacteriaceae is Escherichia coli, Pantoea ananatis, or Enterobacter aerogenes.
 17. The method according to claim 1, wherein the dicarboxylic acid is selected from the group consisting of malic acid, succinic acid, fumaric acid, 2-hydroxyglutaric acid, and α-ketoglutaric acid.
 18. The method according to claim 1, wherein the microorganism has been further modified so that malyl-CoA-producing ability is increased.
 19. The method according to claim 1, wherein the microorganism has been further modified so that α-ketoglutarate synthase activity is increased. 